CD133 epitopes

ABSTRACT

An immunogen includes an isolated peptide that includes the amino sequence of any one of SEQ ID NOs:1-21 with four or fewer amino acid substitutions.

CLAIM OF PRIORITY

This application claims priority to U.S. Patent Application Ser. No.61/176,302, filed on May 7, 2009, the entire contents of which arehereby incorporated by reference.

TECHNICAL FIELD

This invention relates to methods and compositions for the treatment ofcancers.

BACKGROUND

The cell surface marker CD133 (Prominin 1) is expressed by neural stemcells and has been used to select for brain cancer stem cells. Inaddition, CD133 positive cells are highly enriched for cancer stem cellsin colon cancer, hepatocellular carcinoma, prostate cancer, multiplemyeloma, and melanoma.

SUMMARY

This invention is based, in part, on the discovery of peptides of humanCD133 that bind to human leukocyte antigens (HLA) and can stimulateimmune responses. These peptides can be used in immunotherapy ofcancers. Accordingly, compositions for cancer immunotherapy and methodsfor inducing immune responses in cancer patients against tumor antigensare provided herein.

In one aspect, the invention features an immunogen that includes anisolated peptide that includes the amino sequence of any of SEQ IDNOs:1-21 with four or fewer (e.g., three or fewer, two or fewer, one, orzero) amino acid substitutions (e.g., conservative substitutions). Insome embodiments, the immunogen is 800 amino acid residues or fewer(e.g., 700 amino acid residues or fewer, 600 amino acid residues orfewer, 500 amino acid residues or fewer, 400 amino acid residues orfewer, 300 amino acid residues or fewer, 200 amino acid residues orfewer, 150 amino acid residues or fewer, 100 amino acid residues orfewer, 80 amino acid residues or fewer, 60 amino acid residues or fewer,50 amino acid residues or fewer, 40 amino acid residues or fewer, 30amino acid residues or fewer, 20 amino acid residues or fewer, 15 aminoacid residues or fewer, 14 amino acid residues or fewer, 13 amino acidresidues or fewer, 12 amino acid residues or fewer, 11 amino acidresidues or fewer, 10 amino acid residues or fewer, or 9 amino acidresidues). In some embodiments, the immunogen comprises no more than 800consecutive amino acid residues (e.g., no more than 700 amino acidresidues, no more than 600 amino acid residues, no more than 500 aminoacid residues, no more than 400 amino acid residues, no more than 300amino acid residues, no more than 200 amino acid residues, no more than150 amino acid residues, no more than 100 amino acid residues, no morethan 80 amino acid residues, no more than 60 amino acid residues, nomore than 50 amino acid residues, no more than 40 amino acid residues,no more than 30 amino acid residues, no more than 20 amino acidresidues, no more than 15 amino acid residues, no more than 14 aminoacid residues, no more than 13 amino acid residues, no more than 12amino acid residues, no more than 11 amino acid residues, no more than10 amino acid residues, or no more than or 9 amino acid residues) of SEQID NO:26. In some embodiments, the immunogen includes a superagonistvariant of any of SEQ ID NOs:1-21. In some embodiments, the immunogendoes not include the sequence FLLPALIFAV (SEQ ID NO:27).

In another aspect, the invention features compositions that include animmunogen described herein linked to an immunogenic carrier, e.g., aserum albumin, tetanus toxoid, keyhole limpet hemocyanin, dextran, anagonist of a Toll-like receptor (TLR), or a recombinant virus particle.

In another aspect, the invention features polynucleotides that include anucleic acid sequence encoding an immunogen described herein. Thepolynucleotides can include an expression vector, e.g., a plasmid or anonreplicative viral vector (e.g., vaccinia, fowlpox, Venezuelan equineencephalitis virus, adeno-associated virus, and adenovirus). In someembodiments the expression vector is a virus, e.g., an RNA or DNA virus.

In another aspect, the invention features compositions (e.g.,pharmaceutical or vaccine compositions) that include an immunogen orpolynucleotide described herein. The compositions can further include anadjuvant (e.g., complete Freund's adjuvant, incomplete Freund'sadjuvant, Montanide ISA-51, LAG-3, aluminum phosphate, aluminumhydroxide, alum, or saponin), a cytokine (e.g., Interleukin-1 (IL-1),IL-2, IL-7, IL-12, IL-13, IL-15, tumor necrosis factor (TNF), stem cellfactor (SCF), or granulocyte monocyte colony stimulating factor(GM-CSF)), and/or an agonist of a Toll-like receptor (TLR) (e.g., anagonist of TLR-3, TLR-4, TLR-7, or TLR-9). The compositions can includea vehicle, e.g., a liposome (e.g., an emulsion, a foam, a micel, aninsoluble monolayer, a liquid crystal, a phospholipid dispersion, or alamellar layer), an immuno stimulating complex (ISCOM), or aslow-releasing particle.

In a further aspect, the invention features methods of immunization thatinclude administering to a subject an immunogen, polynucleotide, orcomposition described herein in an amount effective to stimulate animmune response (e.g., a therapeutic or prophylactic immune response).The invention also features the use of an immunogen, polynucleotide, orcomposition described herein in the preparation of a medicament forstimulating an immune response. The invention also features the use ofan immunogen, polynucleotide, or composition described herein tostimulate an immune response.

In another aspect, the invention features methods for treating a subjectwith a cancer (e.g., a cancer characterized by tumor cells expressing aclass I MHC molecule). The methods include administering to the subjectan immunogen, polynucleotide, or composition described herein in anamount effective to induce a CTL response to the tumor cells. Theinvention also features the use of an immunogen, polynucleotide, orcomposition described herein in the preparation of a medicament fortreating a subject with a cancer (e.g., a cancer characterized by tumorcells expressing a class I MHC molecule). The invention also featuresthe use of an immunogen, polynucleotide, or composition described hereinfor treating a subject with a cancer (e.g., a cancer characterized bytumor cells expressing a class I MHC molecule).

In a further aspect, the invention features methods for treating asubject with a cancer characterized by tumor cells expressing HLA-A1,HLA-A2, or HLA-A3. The methods include administering to the subjectinduced cytotoxic T lymphocyte (CTLs) in an amount sufficient to destroythe tumor cells through direct lysis or to effect the destruction of thetumor cells indirectly through the elaboration of cytokines, wherein theCTLs are induced by a process that includes inducing a CTL in vitro thatis specific for the tumor cells by contacting a precursor CTL with animmunogen described herein under conditions that generate a CTL responseto the tumor cells. The invention also features the use of an immunogen,polynucleotide, or composition described herein in the preparation of amedicament for treating a subject with a cancer characterized by tumorcells expressing HLA-A1, HLA-A2, or HLA-A3. The invention also featuresthe use of an immunogen, polynucleotide, or composition described hereinfor treating a subject with a cancer characterized by tumor cellsexpressing HLA-A1, HLA-A2, or HLA-A3.

In another aspect, the invention features methods for treating a subjectwith a cancer characterized by tumor cells expressing any class I MHCmolecule. The methods include administering to the subject inducedcytotoxic T lymphocyte (CTLs) in an amount sufficient to destroy thetumor cells through direct lysis or to effect the destruction of thetumor cells indirectly through the elaboration of cytokines, said CTLsare induced by a process comprising inducing a CTL in vitro that isspecific for said tumor cells by contacting a precursor CTL with animmunogen described herein under conditions that generate a CTL responseto the tumor cells.

In a further aspect, the invention features methods for inducing acytotoxic T lymphocyte (CTL) in vitro that is specific for a tumor cellexpressing HLA-A1, HLA-A2, or HLA-A3. The methods include contacting aprecursor CTL with an immunogen described herein under conditions thatgenerate a CTL response to the tumor cells.

In another aspect, the invention features methods for inducing acytotoxic T lymphocyte (CTL) response in vitro that is specific for atumor cell expressing HLA-A1, HLA-A2, or HLA-A3. The methods includecontacting a precursor CTL with a cell that includes a polynucleotidehaving a nucleic acid sequence encoding at least one polypeptide thatincludes an immunogen described herein.

In a further aspect, the invention features methods for treating asubject with a cancer characterized by tumor cells expressing HLA-A1,HLA-A2, or HLA-A3. The methods include administering CTLs induced by amethod described herein in an amount effective to destroy the tumorcells through direct lysis or to effect the destruction of the tumorcells indirectly through the elaboration of cytokines.

The invention also features methods for treating a cancer in a patientthat include administering to the patient a composition comprisingantigen-presenting cells (e.g., dendritic cells), wherein the antigenpresenting cells present on their surface a peptide epitope comprisingthe amino sequence of any of SEQ ID NOs:1-21 with four or fewer (e.g.,three or fewer, two or fewer, one, or zero) amino acid substitutions(e.g., conservative substitutions) or a superagonist variant of any ofSEQ ID NOs:1-21. In some embodiments, the antigen presenting cells(e.g., dendritic cells) acquire the peptide epitopes in vitro byexposure to synthetic peptides having the peptide epitopes. Theinvention also features the use of antigen presenting cells that presenton their surface an immunogen described herein in the preparation of amedicament for treating a subject with cancer. The invention alsofeatures the use of antigen presenting cells that present on theirsurface an immunogen described herein for treating a subject withcancer.

In a further aspect, the invention features methods for preparing a cellvaccine for treating a cancer. The methods include: obtaining bonemarrow derived mononuclear cells from a patient, culturing themononuclear cells in vitro under conditions in which mononuclear cellsbecome adherent to a culture vessel; selecting a subset of themononuclear cells comprising adherent cells; culturing the adherentcells in the presence of one or more cytokines under conditions in whichthe cells differentiate into antigen presenting cells; and culturing theantigen presenting cells in the presence of an immunogen describedherein under conditions in which the cells present the peptides on majorhistocompatibility class I molecules, thereby preparing a cell vaccine.

In any of the above aspects, a cancer or tumor may include one or morecells that express CD133.

In another aspect, the invention features kits that include one or moreimmunogens, polynucleotides, and/or compositions described herein.

A “superagonist” or “superantigen” peptide is a peptide that includesone or more mutations (e.g., one, two, or three amino acid changes,relative to a native (wild type) sequence) and that elicits anantigen-specific immunological response that is more potent than aresponse elicited against a peptide having a native sequence. Forexample, a superagonist peptide stimulates higher levels of IFN-γrelease by antigen-specific T cells, as compared to T cells stimulatedwith the native peptide. The increase in levels of IFN-γ releasestimulated by a superagonist peptide is at least higher than levelsstimulated by a native peptide by a statistically significant amount. Insome embodiments, a superagonist stimulates IFN-γ levels that are atleast 5%, 10%, 25%, 50%, 100%, 200%, or 500% higher than elicited by thenative peptide.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York,N.Y. 2001); March, Advanced Organic Chemistry Reactions, Mechanisms andStructure 5th ed., J. Wiley & Sons (New York, N.Y. 2001); Sambrook andRussel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold SpringHarbor Laboratory Press (Cold Spring Harbor, N.Y. 2001); and Lutz etal., Handbook of Dendritic Cells: Biology, Diseases and Therapies, J.Wiley & Sons (New York, N.Y. 2006), provide one skilled in the art witha general guide to many of the terms used in the present application.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C depict a multiple alignment of human (Hsap, SEQ ID NO:26),chimpanzee (Ptro, SEQ ID NO:30), Rhesus macaque (Mmul, SEQ ID NO:31),rat (Rnor, SEQ ID NO:32), mouse (Mmus, SEQ ID NO:33), dog (Cfam, SEQ IDNO:34), and cattle (Btau, SEQ ID NO:35) CD133 proteins. The multiplealignment was prepared using ClustalW2 (Larkin et al., 2007,Bioinformatics, 23:2947-48). “*”, residues are identical in allsequences; “:”, conserved substitutions; “.”, semi-conservedsubstitutions.

DETAILED DESCRIPTION

The present invention relates to immunogens and immunogeniccompositions, and methods of use thereof, for the prevention, treatment,and/or diagnosis of cancers. Described herein are immunogens thatinclude proteins or polypeptides whose amino acid sequences include oneor more epitopic oligopeptides. In addition, the invention furtherrelates to polynucleotides that can be used to stimulate a CTL responseagainst cancers.

Described herein are specific oligopeptide sequences with amino acidsequences shown in SEQ ID NOs:1-21, which represent epitopic peptides(i.e., immunogenic oligopeptide sequences) of at least about 9-10 aminoacids in length.

CD133 is present in several human cancers (Mizrak et al., 2008, J.Pathol., 214:3-9; Neuzil et al., 2007, Biochem. Biophys. Res. Commun.,355:855-859), including brain cancer, colon cancer, hepatocellularcarcinoma, prostate cancer, multiple myeloma, and melanoma.

An exemplary human CD133 sequence has the following amino acid sequence(SEQ ID NO:26).

(SEQ ID NO: 26) MALVLGSLLLLGLCGNSFSGGQPSSTDAPKAWNYELPATNYETQDSHKAGPIGILFELVHIFLYVVQPRDFPEDTLRKFLQKAYESKIDYDKPETVILGLKIVYYEAGIILCCVLGLLFIILMPLVGYFFCMCRCCNKCGGEMHQRQKENGPFLRKCFAISLLVICIIISIGIFYGFVANHQVRTRIKRSRKLADSNFKDLRTLLNETPEQIKYILAQYNTTKDKAFTDLNSINSVLGGGILDRLRPNIIPVLDEIKSMATAIKETKEALENMNSTLKSLHQQSTQLSSSLTSVKTSLRSSLNDPLCLVHPSSETCNSIRLSLSQLNSNPELRQLPPVDAELDNVNNVLRTDLDGLVQQGYQSLNDIPDRVQRQTTTVVAGIKRVLNSIGSDIDNVTQRLPIQDILSAFSVYVNNTESYIHRNLPTLEEYDSYWWLGGLVICSLLTLIVIFYYLGLLCGVCGYDRHATPTTRGCVSNTGGVFLMVGVGLSFLFCWILMIIVVLTFVFGANVEKLICEPYTSKELFRVLDTPYLLNEDWEYYLSGKLFNKSKMKLTFEQVYSDCKKNRGTYGTLHLQNSFNISEHLNINEHTGSISSELESLKVNLNIFLLGAAGRKNLQDFAACGIDRMNYDSYLAQTGKSPAGVNLLSFAYDLEAKANSLPPGNLRNSLKRDAQTIKTIHQQRVLPIEQSLSTLYQSVKILQRTGNGLLERVTRILASLDFAQNFITNNTSSVIIEETKKYGRTIIGYFEHYLQWIEFSISEKVASCKPVATALDTAVDVFLCSYIIDPLNLFWFGIGKATVFLLPALIFAVKLAKYYRRMDSEDVYDDVETIPMKNMENGNNGYHKDHVYGIHNPVMTSPSQH

The polypeptides forming the immunogens described herein have amino acidsequences that include SEQ ID NOs:1-21 and variants thereof with four orfewer (e.g., three or fewer, two or fewer, one, or zero) amino acidsubstitutions (e.g., conservative substitutions).

Such polypeptides can be of any desired length so long as they haveimmunogenic activity in that they are able, under a given set ofconditions, to elicit in vitro or in vivo the activation of cytotoxic Tlymphocytes (CTLs) (i.e., a CTL response) against a presentation ofCD133 in vitro or in vivo by an antigen presenting cell (APC). Exemplarypolypeptides include those of 800 amino acid residues or fewer (e.g.,700 amino acid residues or fewer, 600 amino acid residues or fewer, 500amino acid residues or fewer, 400 amino acid residues or fewer, 300amino acid residues or fewer, 200 amino acid residues or fewer, 150amino acid residues or fewer, 100 amino acid residues or fewer, 80 aminoacid residues or fewer, 60 amino acid residues or fewer, 50 amino acidresidues or fewer, 40 amino acid residues or fewer, 30 amino acidresidues or fewer, 20 amino acid residues or fewer, 15 amino acidresidues or fewer, 14 amino acid residues or fewer, 13 amino acidresidues or fewer, 12 amino acid residues or fewer, 11 amino acidresidues or fewer, 10 amino acid residues or fewer, or 9 amino acidresidues). The polypeptides forming the immunogens described herein canbe naturally occurring or can be synthesized chemically. Thepolypeptides can include at least one of SEQ ID NOs:1-21.

In some embodiments, an immunogen described herein can a variantsequence such as the counterpart of any of SEQ ID NOs:1-21 from theCD133 protein of an animal species (e.g., chimpanzee, Rhesus macaque,rat, mouse, dog, or cattle). A counterpart peptide can be identified byaligning the human and animal CD133 proteins (e.g., as shown in FIGS.1A-C) and selecting the sequence from the animal protein that alignswith the portion of the human sequence corresponding to the peptide ofinterest. For example, SEQ ID NO:11 and its animal counterparts areshown in underscore in FIG. 1C. In some instances, the counterpartsequence immunogen may have more than four amino acid differences ascompared to the human sequence.

Oligopeptides as disclosed herein may themselves be prepared by methodswell known to those skilled in the art. See, e.g., Grant, G. A.,Synthetic Peptides: A User's Guide, 1992, W. H. Freeman and Company, NewYork; Coligan, J. E. et al, Current Protocols in Protein Science, 1999,John Wiley & Sons, Inc., New York.

Besides the sequences of SEQ ID NOs:1-21, the proteins and polypeptidesforming the immunogens described herein can also include one or moreother immunogenic amino acid stretches known to be associated withcancers, and which may stimulate a CTL response whereby the immunogenicpeptides associate with HLA-A1, HLA-A2, HLA-A3, HLA-A1/A11, HLAsupertypes, or any class I MHC (i.e., MHC-1) molecule.

The oligopeptides and polypeptides described herein can be derived byfractionation of naturally occurring proteins by methods such asprotease treatment, or they can be produced by recombinant or syntheticmethodologies that are well known and clear to the skilled artisan. See,e.g., Ausubel, F. M. et al, Current Protocols in Molecular Biology,1999, John Wiley & Sons, Inc., New York; Coligan, J. E. et al, CurrentProtocols in Protein Science, 1999, John Wiley & Sons, Inc., New York;Molecular Cloning: A Laboratory Manual, 1989, Cold Spring HarborLaboratory Press, Cold Spring Harbor. The polypeptide can include arecombinant or synthetic polypeptide that includes at least one of SEQID NOs:1-21, which sequences can also be present in multiple copies.Thus, oligopeptides and polypeptides disclosed herein can have one, two,three, or more such immunogenic peptides within the amino acid sequenceof said oligopeptides and polypeptides, and said immunogenic peptides,or epitopes, can be the same or can be different, or can have any numberof such sequences, wherein some of them are identical to each other inamino acid sequence while others within the same polypeptide sequenceare different from each other and said epitopic sequences can occur inany order within said immunogenic polypeptide sequence. The location ofsuch sequences within the sequence of a polypeptide forming an immunogendescribed herein can affect relative immunogenic activity. In addition,immunogens described herein can include more than one protein comprisingthe amino acid sequences disclosed herein. Such polypeptides can be partof a single composition or can themselves be covalently ornon-covalently linked to each other.

The immunogenic peptides described herein can also be linked directlyto, or through a spacer or linker to: an immunogenic carrier such asserum albumin, tetanus toxoid, keyhole limpet hemocyanin, dextran, or arecombinant virus particle; a Toll-like receptor (TLR) agonist; animmunogenic peptide known to stimulate a T helper cell type immuneresponse; a cytokine such as interferon gamma or GM-CSF; a targetingagent such as an antibody or receptor ligand; a stabilizing agent suchas a lipid; or a conjugate of a plurality of epitopes to a branchedlysine core structure, such as the so-called “multiple antigenicpeptide” described in Posneft et al., 1988, J. Biol. Chem.,263:1719-1725; a compound such as polyethylene glycol to increase thehalf life of the peptide; or additional amino acids such as a leader orsecretory sequence, or a sequence employed for the purification of themature sequence. Spacers and linkers typically include relatively small,neutral molecules, such as amino acids and which are substantiallyuncharged under physiological conditions. Such spacers are typicallyselected from the group of nonpolar or neutral polar amino acids, suchas glycine, alanine, serine and other similar amino acids. Such optionalspacers or linkers need not include the same residues and thus can beeither homo- or hetero-oligomers. When present, such linkers willcommonly be of length at least one or two, commonly 3, 4, 5, 6, andpossibly as much as 10 or even up to 20 residues (in the case of aminoacids). In addition, such linkers need not be composed of amino acidsbut any oligomeric structures will do as well so long as they providethe correct spacing so as to optimize the desired level of immunogenicactivity of the immunogens described herein. The immunogen can thereforetake any form that is capable of eliciting a CTL response.

In addition, the immunogenic peptides described herein can be part of animmunogenic structure via attachments other than conventional peptidebonds. Thus, any manner of attaching the peptides to an immunogendescribed herein, such as an immunogenic polypeptide, could provide animmunogenic structure. Thus, immunogens, such as proteins, oligopeptidesand polypeptides, are structures that contain the peptides disclosed,but such immunogenic peptides may not necessarily be attached thereto bythe conventional means of using ordinary peptide bounds. The immunogensdescribed herein simply contain such peptides as part of their makeup,but how such peptides are to be combined to form the final immunogen isleft to the talent and imagination of the user and is in no wayrestricted or limited by the disclosure contained herein.

It should be appreciated that an immunogen described herein can consistonly of a peptide of SEQ ID NOs:1-21 (or a variant thereof), or includea peptide of SEQ ID NOs:1-21 (or a variant thereof), or include aplurality of peptides selected from SEQ ID NOs:1-21 (or one or morevariants thereof), or include a polypeptide that itself includes one ormore of the epitopic peptides of SEQ ID NOs:1-21 (or one or morevariants thereof). In some embodiments, an immunogen, composition, orkit described herein can further include or exclude a polypeptide,epitope, or other antigenic composition described in US 2007/0020297; US2008/0206296; US 2008/0311142; or WO 2010/028066, all of which areincorporated by reference herein.

Modified Peptides

The peptides that are naturally processed and bound to a class I MHCmolecule, and which are recognized by a tumor-specific CTL, are notnecessarily the optimal peptides for stimulating a CTL response. See,for example, Parkhurst et al., 1996, J. Immunol., 157:2539-48; Rosenberget al., 1998, Nat. Med., 4:321-32. Thus, there can be utility inmodifying a peptide, such that it more readily or effectively induces aCTL response. Typically, peptides can be modified at two types ofpositions. The peptides can be modified at amino acid residues that arepredicted to interact with the class I MHC molecule, in which case thegoal is to create a peptide that has a higher affinity for the class IMHC molecule than does the original peptide. The peptides can also bemodified at amino acid residues that are predicted to interact with theT cell receptor on the CTL, in which case the goal is to create apeptide that has a higher affinity for the T cell receptor than does theoriginal peptide. Both of these types of modifications can result in avariant peptide that is related to an original peptide, but which isbetter able to induce a CTL response than is the original peptide. Asused herein, the term “original peptide” means an oligopeptide with theamino acid sequence selected from SEQ ID NOs:1-21.

The original peptides disclosed herein can be modified by thesubstitution of one or more residues at different, possibly selective,sites within the peptide chain. Such substitutions can be of aconservative nature, for example, where one amino acid is replaced by anamino acid of similar structure and characteristics, such as where ahydrophobic amino acid is replaced by another hydrophobic amino acid.Even more conservative would be replacement of amino acids of the sameor similar size and chemical nature, such as where leucine is replacedby isoleucine. In studies of sequence variations in families ofnaturally occurring homologous proteins, certain amino acidsubstitutions are more often tolerated than others, and these often showcorrelation with similarities in size, charge, polarity, andhydrophobicity between the original amino acid and its replacement, andsuch is the basis for defining “conservative substitutions.”

Conservative substitutions are defined herein as exchanges within one ofthe following five groups: Group 1—small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2—polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3—polar,positively charged residues (His, Arg, Lys); Group 4—large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 4—large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics, but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly nonconservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character, or vice versa.

Such substitutions can also involve structures other than the commonL-amino acids. Thus, D-amino acids might be substituted for the L-aminoacids commonly found in the antigenic peptides described herein and yetstill be encompassed by the present disclosure. In addition, amino acidspossessing non-standard R groups (i.e., R groups other than those foundin the 20 common amino acids of natural proteins) can also be used forsubstitution purposes to produce immunogens and immunogenicpolypeptides.

Based on cytotoxicity assays, a substituted epitopic peptide isconsidered substantially identical to the reference peptide if it has atleast 10% of the antigenic activity of the reference peptide as definedby the ability of the substituted peptide to reconstitute the epitoperecognized by a CTL in comparison to the reference peptide. Thus, whencomparing the lytic activity in the linear portion of theeffector:target curves with equimolar concentrations of the referenceand substituted peptides, the observed percent specific killing of thetarget cells incubated with the substituted peptide should be equal tothat of the reference peptide at an effector:target ratio that is nogreater than 10-fold above the reference peptide effector:target ratioat which the comparison is being made.

Thus, the epitopes described herein can be identical to naturallyoccurring tumor-associated or tumor-specific epitopes or can includeepitopes that differ by no more than 4 residues from the referencepeptide, as long as they have substantially identical antigenicactivity.

Preparation of Immunogenic Peptides and Structures

The immunogenic peptides and polypeptides described herein can beprepared synthetically, by recombinant DNA technology, or they can beisolated from natural sources such as tumor cells expressing theoriginal protein product.

The polypeptides and oligopeptides disclosed herein can be synthesizedin solution or on a solid support in accordance with conventionaltechniques. Various automated peptide synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Grant, G. A., Synthetic Peptides: A User's Guide, 1992, W. H.Freeman and Company, New York; Coligan, J. E. et al, Current Protocolsin Protein Science, 1999, John Wiley & Sons, Inc., New York. Fragmentsof polypeptides described herein can also be synthesized asintermediates in the synthesis of a larger polypeptide.

Recombinant DNA technology can be employed wherein a nucleotide sequencethat encodes an immunogenic peptide or polypeptide of interest isinserted into an expression vector, transformed or transfected into anappropriate host cell, and cultivated under conditions suitable forexpression. These procedures are well known in the art to the skilledartisan, as described in, e.g., Coligan, J. E. et al, Current Protocolsin Immunology, 2006, John Wiley & Sons, Inc., New York; Ausubel, F. M.et al, Current Protocols in Molecular Biology, 1999, John Wiley & Sons,Inc., New York; Molecular Cloning: A Laboratory Manual, 1989, ColdSpring Harbor Laboratory Press, Cold Spring Harbor. Thus, recombinantlyproduced peptides or polypeptides can be used as the immunogensdescribed herein.

The coding sequences for peptides of the length contemplated herein canalso be synthesized on commercially available automated DNA synthesizersusing protocols that are well know in the art. See for example, Grant,G. A., Synthetic Peptides: A User's Guide, 1992, W. H. Freeman andCompany, New York; Coligan, J. E. et al, Current Protocols in ProteinScience, 1999, John Wiley & Sons, Inc., New York. The coding sequencescan also be modified such that a peptide or polypeptide will be producedthat incorporates a desired amino acid substitution. The coding sequencecan be provided with appropriate linkers, be ligated into suitableexpression vectors that are commonly available in the art, and theresulting DNA or RNA molecule can be transformed or transfected intosuitable hosts to produce the desired fusion protein. A number of suchvectors and suitable host systems are available, and their selection isleft to the skilled artisan. For expression of the fusion proteins, thecoding sequence will be provided with operably linked start and stopcodons, promoter and terminator regions, and a replication system toprovide an expression vector for expression in the desired host cell.For example, promoter sequences compatible with bacterial hosts areprovided in plasmids containing convenient restriction sites forinsertion of the desired coding sequence. The resulting expressionvectors are transformed into suitable bacterial hosts. Yeast, insect,and mammalian host cells can also be used, employing suitable vectorsand control sequences.

Host cells can be genetically engineered (e.g., transduced, transformed,or transfected) with the vectors described herein which can be, forexample, a cloning vector or an expression vector. The vector can be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

The present invention also includes recombinant constructs comprisingone or more of the sequences as broadly described above. The constructsinclude a vector, such as a plasmid or viral vector, into which asequence described herein has been inserted, in a forward or reverseorientation. In a preferred aspect of this embodiment, the constructfurther includes regulatory sequences, including, for example, apromoter, operably linked to the sequence. Large numbers of suitablevectors and promoters are known to those of skill in the art, and arecommercially available.

Host Cells

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation. See, e.g., Ausubel, F. M. et al,Current Protocols in Molecular Biology, 1999, John Wiley & Sons, Inc.,New York; Molecular Cloning: A Laboratory Manual, 1989, Cold SpringHarbor Laboratory Press, Cold Spring Harbor. Such cells can routinely beutilized for assaying CTL activity by having said geneticallyengineered, or recombinant, host cells express the immunogenic peptidesdescribed herein.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,1981, Cell, 23:175, and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will include an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnon-transcribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites can be used to provide the requirednontranscribed genetic elements.

The polypeptides can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature peptides and proteins. Highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

Antigen-Presenting Cells

Antigen presenting cells that are to be used to stimulate a CTL responseare typically incubated with a peptide of an optimal length, for examplea nonapeptide, that allows for direct binding of the peptide to theclass I MHC molecule without additional processing. Larger oligopeptidesand polypeptides are generally ineffective in binding to class I MHCmolecules as they are not efficiently processed into an appropriatelysized peptide in the extracellular milieu. A variety of approaches areknown in the art, however, that allow oligopeptides and polypeptides tobe exogenously acquired by a cell, which then allows for theirsubsequent processing and presentation by a class I MHC molecule.Representative, but non-limiting examples of such approaches includeelectroporation of the molecules into the cell (Harding, 1992, Eur. J.Immunol., 22:1865-69), encapsulation of the molecules in liposomes thatare fused to the cells of interest (Reddy et al., 1991, J. Immunol.Methods, 141:157-163), or osmotic shock in which the molecules are takenup via pinocytosis (Moore et al., 1988, Cell, 54:777-785). Thus,oligopeptides and polypeptides that include one or more of the peptidesdescribed herein can be provided to antigen presenting cells in such afashion that they are delivered to the cytoplasm of the cell, and aresubsequently processed to allow presentation of the peptides.

Antigen presenting cells suitable for stimulating an in vitro CTLresponse that is specific for one or more of the peptides describedherein can also be prepared by introducing polynucleotide vectorsencoding the sequences into the cells. These polynucleotides can bedesigned such that they express only a single peptide, multiplepeptides, or even a plurality of peptides. A variety of approaches areknown in the art that allow polynucleotides to be introduced andexpressed in a cell, thus providing one or more peptides describedherein to the class I MHC molecule binding pathway. Representative, butnon-limiting examples of such approaches include the introduction ofplasmid DNA through particle-mediated gene transfer or electroporation(Tuting et al., 1998, J. Immunol., 160:1139-47), or the transduction ofcells with an adenovirus expressing the polynucleotide of interest(Perez-Diez et al., 1998, Cancer Res., 58:5305-09). Thus,oligonucleotides that code for one or more of the peptides describedherein can be provided to antigen presenting cells in such a fashionthat the peptides associate with class I MHC molecules and are presentedon the surface of the antigen presenting cell, and consequently areavailable to stimulate a CTL response.

In certain embodiments, the methods described herein include a methodfor inducing a CTL response in vitro that is specific for a tumor cellexpressing a molecule from A1, A2, or A3 supertypes (A11 is a member ofthe A3 supertype), whereby the method includes contacting a CTLprecursor lymphocyte with an antigen presenting cell that has bound toan immunogen comprising one or more of the peptides disclosed herein.

In specific embodiments, the methods described herein include a methodfor inducing a CTL response in vitro that is specific for a tumor cellexpressing a molecule from A1, A2, or A3 supertypes, whereby the methodincludes contacting a CTL precursor lymphocyte with an antigenpresenting cell that has exogenously acquired an immunogenicoligopeptide or polypeptide that includes one or more of the peptidesdisclosed according to the invention.

A yet additional embodiment described herein is directed to a processfor inducing a CTL response in vitro that is specific for a tumor cellexpressing a molecule from A1, A2, or A3 supertypes, comprisingcontacting a CTL precursor lymphocyte with an antigen presenting cellthat is expressing a polynucleotide coding for a polypeptide describedherein, and wherein said polynucleotide is operably linked to apromoter.

A variety of techniques exist for assaying the activity of CTL. Thesetechniques include the labeling of target cells with radionuclides suchas Na₂ ⁵¹CrO₄ or ³H-thymidine, and measuring the release or retention ofthe radionuclides from the target cells as an index of cell death. Suchassays are well-known in the art. Alternatively, CTL are known torelease a variety of cytokines when they are stimulated by anappropriate target cell, such as a tumor cell expressing the relevantclass I MHC molecule and the corresponding peptide. Non-limitingexamples of such cytokines include IFN-γ, TNF-α, and GM-CSF. Assays forthese cytokines are well known in the art. Methodology for measuringboth target cell death and cytokine release as a measure of CTLreactivity are given in Coligan, J. E. et al. (Current Protocols inImmunology, 1999, John Wiley & Sons, Inc., New York).

After expansion of the antigen-specific CTLs, the latter can then betransferred back into the patient, where they will destroy theirspecific target cell. The utility of such adoptive transfer isdemonstrated in North et al. (199, Infect. Immun., 67:2010-12) andRiddell et al. (1992, Science, 257:238-241). In determining the numberof cells to reinfuse, the skilled physician will be guided by the totalnumber of cells available, the activity of the CTL as measured in vitro,and the condition of the patient. Typically, about 1×10⁶ to about 1×10¹²(e.g., about 1×10⁸ to about 1×10¹¹ or about 1×10⁹ to about 1×10¹⁰)peptide-specific CTL are infused. Methods for reinfusing T cells into apatient are well known and exemplified in U.S. Pat. No. 4,844,893 toHonski, et al., and U.S. Pat. No. 4,690,915 to Rosenberg.

The peptide-specific CTL can be purified from the stimulator cells priorto infusion into the patient. For example, monoclonal antibodiesdirected toward the cell surface protein CD8, present on CTL, can beused in conjunction with a variety of isolation techniques such asantibody panning, flow cytometric sorting, and magnetic bead separationto purify the peptide-specific CTL away from any remaining non-peptidespecific lymphocytes or from the stimulator cells. These methods arewell known in the art. It should be appreciated that generation ofpeptide-specific CTL in this manner obviates the need for stimulatingthe CTL in the presence of tumor. Thus, there is no chance ofinadvertently reintroducing tumor cells into the patient.

Thus, one embodiment of the present invention relates to a process fortreating a subject who has cancer characterized by tumor cellsexpressing complexes of a molecule from A1, A2, or A3 supertypes, forexample, HLA-A1, HLA-A2, HLA-A3, or HLAA11, whereby CTLs produced invitro according to the methods described herein are administered in anamount sufficient to destroy the tumor cells through direct lysis or toeffect the destruction of the tumor cells indirectly through theelaboration of cytokines.

Another embodiment of the present invention is directed to a process fortreating a subject with cancer characterized by tumor cells expressingany class I MHC molecule and an epitope of SEQ ID NOs:1-21, whereby theCTLs are produced in vitro and are specific for the epitope or originalprotein and are administered in an amount sufficient to destroy thetumor cells through direct lysis or to effect the destruction of thetumor cells indirectly through the elaboration of cytokines.

The ex vivo generated CTL can be used to identify and isolate the T cellreceptor molecules specific for the peptide. The genes encoding thealpha and beta chains of the T cell receptor can be cloned into anexpression vector system and transferred and expressed in naive T cellsfrom peripheral blood, T cells from lymph nodes, or T lymphocyteprogenitor cells from bone marrow. These T cells, which would then beexpressing a peptide-specific T cell receptor, would then haveanti-tumor reactivity and could be used in adoptive therapy of cancers.

Screening and Diagnostic Methods

In addition to their use for therapeutic or prophylactic purposes, theimmunogenic peptides described herein are useful as screening anddiagnostic agents. Thus, the immunogenic peptides described herein,together with modern techniques of gene screening, make it possible toscreen patients for the presence of genes encoding such peptides oncells obtained by biopsy of tumors detected in such patients. Forexample, patients can be screened using nucleic acids or antibodies todetect the expression of CD133. The results of such screening can helpdetermine the efficacy of proceeding with the regimen of treatmentdisclosed herein using the immunogens described herein.

Alternatively, the immunogenic peptides disclosed herein, as well asfunctionally similar homologs thereof, can be used to screen a samplefor the presence of CTLs that specifically recognize the correspondingepitopes. The lymphocytes to be screened in this assay will normally beobtained from the peripheral blood, but lymphocytes can be obtained fromother sources, including lymph nodes, spleen, tumors, and pleural fluid.The peptides described herein can then be used as a diagnostic tool toevaluate the efficacy of the immunotherapeutic treatments disclosedherein. Thus, the in vitro generation of CTL as described above would beused to determine if patients are likely to respond to the peptide invivo. Similarly, the in vitro generation of CTL could be done withsamples of lymphocytes obtained from the patient before and aftertreatment with the peptides. Successful generation of CTL in vivo shouldthen be recognized by a correspondingly easier ability to generatepeptide-specific CTL in vitro from lymphocytes obtained followingtreatment in comparison to those obtained before treatment.

The oligopeptides described herein, such as SEQ ID NOs:1-21, can also beused to prepare multimers (e.g., dimers, tetramers, or pentamers), whichcan be used, e.g., in conjunction with flow cytometry, to quantitate thefrequency of peptide-specific CTL that are present in a sample oflymphocytes from an individual. For example, class I MHC moleculescomprising peptides of SEQ ID NOs:1-21, could be combined to formtetramers as exemplified in U.S. Pat. No. 5,635,363. The multimers(e.g., tetramers) can be used in monitoring the frequency of CTLs in theperipheral blood, lymph nodes, or tumor mass of an individual undergoingimmunotherapy with the peptides, proteins, or polynucleotides describedherein, and it would be expected that successful immunization would leadto an increase in the frequency of the peptide-specific CTL. Adescription of peptide tetramers and methods of using them can be foundin Coligan et al, Current Protocols in Immunology, 2006, John Wiley &Sons, Inc., New York.

Methods of Therapy

A vaccine can include one or more of the polypeptides or fragmentsthereof described herein, or a composition, or pool, of immunogenicpeptides disclosed herein. Two or more polypeptides and/or fragmentsthereof can be used as a physical mixture or as a fusion. The fusionfragment or fusion polypeptide can be produced, for example, byrecombinant techniques or by the use of appropriate linkers for fusingpreviously prepared polypeptides or fragments.

The immunogenic molecules described herein, including vaccinecompositions, can be utilized according to the methods described hereinfor purposes of inhibiting, suppressing, or treating diseases causingthe expression of the immunogenic peptides disclosed herein, such aswhere the antigen is being expressed by tumor cells. As used inaccordance with the present application, the term “inhibiting” relatesto a process of prophylaxis in which an animal, especially a mammal, andmost especially a human, is exposed to an immunogen described hereinprior to the induction or onset of the disease process. This could bedone where an individual has a genetic pedigree indicating apredisposition toward occurrence of the disease condition to beprevented. For example, this might be true of an individual whoseancestors show a predisposition toward certain types of cancer.Alternatively, the immunogen could be administered to the generalpopulation as is frequently done for infectious diseases.

The term “suppression” is often used to describe a scenario wherein thedisease process has already begun, but obvious symptoms of saidcondition have yet to be realized. Thus, the cells of an individual mayhave become cancerous, but no outside signs of the disease have yet beenclinically recognized. The term prophylaxis is used herein to encompassboth inhibition and suppression. Conversely, the term “treatment” isused herein to mean the clinical application of agents to combat analready existing condition whose clinical presentation has already beenrealized in a patient. This would typically occur where an individualhas already been diagnosed as having a tumor.

As used herein, the term “cancer” refers to cells having the capacityfor autonomous growth, i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth. Hyperproliferativeand neoplastic disease states may be categorized as pathologic, i.e.,characterizing or constituting a disease state, or may be categorized asnon-pathologic, i.e., a deviation from normal but not associated with adisease state. In general, a cancer will be associated with the presenceof one or more tumors, i.e., abnormal cell masses. The term “tumor” ismeant to include all types of cancerous growths or oncogenic processes,metastatic tissues or malignantly transformed cells, tissues, or organs,irrespective of histopathologic type or stage of invasiveness.“Pathologic hyperproliferative” cells occur in disease statescharacterized by malignant tumor growth.

Tumors include malignancies of the various organ systems, such asaffecting lung, breast, thyroid, lymphoid, gastrointestinal, andgenito-urinary tract, as well as adenocarcinomas which includemalignancies such as most colon cancers, renal-cell carcinoma, prostatecancer and/or testicular tumors, non-small cell carcinoma of the lung,hepatocellular cancer, cancer of the small intestine and cancer of theesophagus. The term “carcinoma” is art recognized and refers tomalignancies of epithelial or endocrine tissues including respiratorysystem carcinomas, gastrointestinal system carcinomas, genitourinarysystem carcinomas, testicular carcinomas, breast carcinomas, prostaticcarcinomas, endocrine system carcinomas, and melanomas. In someembodiments, the disease is renal carcinoma or melanoma. Exemplarycarcinomas include those forming from tissue of the cervix, prostate,breast, head and neck, colon and ovary. The term also includescarcinosarcomas, e.g., which include malignant tumors composed ofcarcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to acarcinoma derived from glandular tissue or in which the tumor cells formrecognizable glandular structures. The term “sarcoma” is art recognizedand refers to malignant tumors of mesenchymal derivation.

Additional examples of cancers that can be treated using the methods andcompositions described herein include brain and nervous system cancers,including, but not limited to, gliomas, glioblastomas, glioblastomamultiforme (GBM), oligodendrogliomas, primitive neuroectodermal tumors,low, mid and high grade astrocytomas, ependymomas (e.g., myxopapillaryependymoma papillary ependymoma, subependymoma, anaplastic ependymoma),oligodendrogliomas, medulloblastomas, meningiomas, pituitary adenomas,neuroblastomas, neurofibromas, malignant peripheral nerve sheath tumors,schwannomas, and craniopharyngiomas.

Additional examples of proliferative disorders include hematopoieticneoplastic disorders. As used herein, the term “hematopoietic neoplasticdisorders” includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. For example, the diseases arisefrom poorly differentiated acute leukemias, e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus (1991) Crit Rev. inOncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

It is understood that the suitable dosage of an immunogen describedherein will depend upon the age, sex, health, and weight of therecipient, the kind of concurrent treatment, if any, the frequency oftreatment, and the nature of the effect desired. However, the mostpreferred dosage can be tailored to the individual subject, asdetermined by the researcher or clinician. The total dose required forany given treatment will commonly be determined with respect to astandard reference dose as set by a manufacturer, such as is commonlydone with vaccines, such dose being administered either in a singletreatment or in a series of doses, the success of which will depend onthe production of a desired immunological result (i.e., successfulproduction of a CTL-mediated response to the antigen, which responsegives rise to the inhibition and/or treatment desired). Thus, theoverall administration schedule must be considered in determining thesuccess of a course of treatment and not whether a single dose, given inisolation, would or would not produce the desired immunologicallytherapeutic result or effect.

The therapeutically effective amount of a composition containing one ormore of the immunogens described herein, is an amount sufficient toinduce an effective CTL response to inhibit or arrest diseaseprogression. Thus, this dose will depend, among other things, on theidentity of the immunogens used, the nature of the disease condition,the severity of the disease condition, the extent of any need to preventsuch a condition where it has not already been detected, the manner ofadministration dictated by the situation requiring such administration,the weight and state of health of the individual receiving suchadministration, and the sound judgment of the clinician or researcher.Thus, for purposes of prophylactic or therapeutic administration,effective amounts would generally lie within the range of from 1.0 μg toabout 5,000 μg of peptide for a 70 kg patient, followed by boostingdosages of from about 1.0 μg to about 1,000 μg of peptide pursuant to aboosting regimen over days, weeks or months, depending on therecipient's response and as necessitated by subsequent monitoring ofCTL-mediated activity within the bloodstream. Of course, such dosagesare to be considered only a general guide and, in a given situation, theactual dosage can exceed such suggested dosage regimens where theclinician believes that the recipient's condition warrants a moreaggressive administration schedule. The efficacy of administeringadditional doses, and of increasing or decreasing the interval, can bere-evaluated on a continuing basis, in view of the recipient'simmunocompetence (for example, the level of CTL activity with respect totumor-associated or tumor-specific antigens).

For such purposes, the immunogenic compositions described herein can beused against a disease condition such as cancer by administration to anindividual by a variety of routes. The compositions can be administeredparenterally or orally, and, if parenterally, either systemically ortopically. Parenteral routes include subcutaneous, intravenous,intradermal, intramuscular, intraperitoneal, intranasal, transdermal, orbuccal routes. One or more such routes can be employed. Parenteraladministration can be, for example, by bolus injection or by gradualperfusion over time.

Typically, vaccines are prepared as injectables, in the form of aqueoussolutions or suspensions. Vaccines in an oil base are also well knownsuch as for inhaling. Solid forms that are dissolved or suspended priorto use can also be formulated. Pharmaceutical carriers, diluents andexcipients are generally added that are compatible with the activeingredients and acceptable for pharmaceutical use. Examples of suchcarriers include, but are not limited to, water, saline solutions,dextrose, or glycerol. Combinations of carriers can also be used. Thesecompositions can be sterilized by conventional, well known sterilizationtechniques including sterile filtration. The resulting solutions can bepackaged for use as is, or the aqueous solutions can be lyophilized, thelyophilized preparation being combined with sterile water beforeadministration. Vaccine compositions can further incorporate additionalsubstances to stabilize pH, or to function as adjuvants, wetting agents,or emulsifying agents, which can serve to improve the effectiveness ofthe vaccine.

The concentration of the CTL stimulatory peptides described herein inpharmaceutical formulations are subject to wide variation, includinganywhere from less than 0.01% by weight to as much as 50% or more.Factors such as volume and viscosity of the resulting composition mustalso be considered. The solvents, or diluents, used for suchcompositions include water, dimethylsulfoxide, PBS (phosphate bufferedsaline), or saline itself, or other possible carriers or excipients.

The immunogens described herein can also be contained in artificiallycreated structures such as liposomes, ISCOMS, slow-releasing particles,and other vehicles which increase the immunogenicity and/or half-life ofthe peptides or polypeptides in serum. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. Liposomes for use in themethods and compositions described herein are formed from standardvesicle-forming lipids which generally include neutral and negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally determined by considerations such as liposomesize and stability in the blood. A variety of methods are available forpreparing liposomes as reviewed, for example, by Coligan, J. E. et al,Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., NewYork and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.Liposomes containing the peptides or polypeptides described herein canbe directed to the site of lymphoid cells where the liposomes thendeliver the selected immunogens directly to antigen presenting cells.Targeting can be achieved by incorporating additional molecules such asproteins or polysaccharides into the outer membranes of said structures,thus resulting in the delivery of the structures to particular areas ofthe body, or to particular cells within a given organ or tissue. Suchtargeting molecules can include a molecule that binds to receptor onantigen presenting cells. For example an antibody that binds to CD80could be used to direct liposomes to dendritic cells.

The immunogens described herein can also be administered as solidcompositions. Conventional nontoxic solid carriers includingpharmaceutical grades of mannitol, lactose, starch, magnesium,cellulose, glucose, sucrose, sodium saccharin, and the like. Such solidcompositions will often be administered orally, whereby apharmaceutically acceptable nontoxic composition is formed byincorporating the peptides and polypeptides described herein with any ofthe carriers listed above. Generally, such compositions will contain10-95% active ingredient, and more preferably 25-75% active ingredient.

Aerosol administration is also an alternative, requiring only that theimmunogens be properly dispersed within the aerosol propellant. Typicalpercentages of the peptides or polypeptides described herein are0.01%-20% by weight, e.g., 1%-10%. The use of a surfactant to properlydisperse the immunogen may be required. Representative surfactantsinclude the esters or partial esters of fatty acids containing from 6 to22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic,linoleic, linolenic, olesteric and oleic acids with an aliphaticpolyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixedor natural glycerides can be employed. The surfactant can constitute0.1-20% by weight of the composition, e.g., 0.25-5%. Typical propellantsfor such administration can include esters and similar chemicals but areby no means limited to these. A carrier, such as lecithin, forintranasal delivery can also be included.

The peptides and polypeptides described herein can also be deliveredwith an adjuvant. Adjuvants include, but are not limited to, Toll-likereceptor (TLR) agonists, Bacillus Calmette Guem (BCG), complete orincomplete Freund's adjuvant, a cytosine guanine oligodeoxynucleotide(CpG-ODN), Montanide ISA-51, Activation Gene-3 (LAG-3), aluminumphosphate, aluminum hydroxide, alum, and saponin. Adjuvant effects canalso be obtained by administering one or more cytokines along with theimmunogens described herein. These cytokines include, but are notlimited to IL-1, IL-2, IL-7, IL-12, IL-13, IL-15, IL-18, and GM-CSF.Exemplary TLR agonists are described in Ghosh et al., 2006, Cell.Immunol., 243:48-57 and Lippincott's Illustrated Reviews: Immunology,Lippincott Williams & Wilkins; (Jul. 1, 2007), ISBN-10: 0781795435, page17.

The peptides and polypeptides described herein can also be added toprofessional antigen presenting cells such as dendritic cells that havebeen prepared ex vivo. For example, the dendritic cells could beprepared from CD34 positive stem cells from the bone marrow, or theycould be prepared from CD14 positive monocytes obtained from theperipheral blood. The dendritic cells are generated ex vivo usingcytokines such as GM-CSF, IL-3, IL-4, TNF, and SCF. The cultured DC arethen pulsed with peptides at various concentrations using standardmethods that are well known in the art. The peptide-pulsed dendriticcells can then be administered either intravenously, subcutaneously, orintradermally, and the immunization can also include cytokines such asIL-2 or IL-12.

An antigen presenting cell (APC)-based cancer vaccine can be deliveredto a patient or test animal by any suitable delivery route, which caninclude injection, infusion, inoculation, direct surgical delivery, orany combination thereof. In some embodiments, the cancer vaccine isadministered to a human in the deltoid region or axillary region. Forexample, the vaccine is administered into the axillary region as anintradermal injection. In other embodiments, the vaccine is administeredintravenously.

An appropriate carrier for administering APCs can be selected by one ofskill in the art by routine techniques. For example, the pharmaceuticalcarrier can be a buffered saline solution, e.g., cell culture media, andcan include DMSO for preserving cell viability.

The quantity of APCs appropriate for administration to a patient as acancer vaccine can be based upon a variety of factors, as can theformulation of the vaccine itself. Some of these factors include thephysical characteristics of the patient (e.g., age, weight, and sex),the physical characteristics of the tumor (e.g., location, size, rate ofgrowth, and accessibility), and the extent to which other therapeuticmethodologies (e.g., chemotherapy, and beam radiation therapy) are beingimplemented in connection with an overall treatment regimen.Notwithstanding the variety of factors one should consider inimplementing the methods described herein to treat a disease condition,a mammal can be administered with from about 10⁵ to about 10⁸ APCs(e.g., 10⁷ APCs) in from about 0.05 mL to about 2 mL solution (e.g.,saline) in a single administration. Additional administrations can becarried out, depending upon the above-described and other factors, suchas the severity of tumor pathology. In one embodiment, from about one toabout five administrations of about 10⁶ APCs is performed at two-weekintervals.

APC vaccination can be accompanied by other treatments. For example, apatient receiving APC vaccination can also be receiving chemotherapy,radiation, and/or surgical therapy concurrently. Methods of treatingcancer using APC vaccination in conjunction with chemotherapy aredescribed in Wheeler et al., US Pat. Pub. No. 2007/0020297, thedisclosure of which is incorporated herein by reference in its entirety.In some embodiments, a patient receiving DC vaccination has alreadyreceived chemotherapy, radiation, and/or surgical treatment for thecancer. In one embodiment, a patient receiving DC vaccination is treatedwith a COX-2 inhibitor, as described in Yu and Akasaki, WO 2005/037995and US 2008/0199484, the disclosure of each being incorporated herein byreference in its entirety.

The present invention is also directed to a vaccine in which animmunogen described herein is delivered or administered in the form of apolynucleotide encoding a polypeptide or fragment as disclosed herein,whereby the peptide or polypeptide or fragment is produced in vivo. Thepolynucleotide can be included in a suitable expression vector andcombined with a pharmaceutically acceptable carrier. For example, thepeptides or polypeptides could be expressed in plasmid DNA andnonreplicative viral vectors such as vaccinia, fowlpox, Venezuelanequine encephalitis virus, adenovirus, or other RNA or DNA viruses.These examples are meant to be illustrative only and should not beviewed as limiting. A wide variety of other vectors is available and areapparent to those skilled in the art from the description given herein.In this approach, a portion of the nucleotide sequence of the viralvector is engineered to express the peptides or polypeptides describedherein. Vaccinia vectors and methods useful in immunization protocolsare described in U.S. Pat. No. 4,722,848, the disclosure of which isincorporated herein by reference in its entirety.

Regardless of the nature of the composition given, additionaltherapeutic agents can also accompany the immunogens described herein.Thus, for purposes of treating tumors, compositions containing theimmunogens disclosed herein can, in addition, contain other antitumorpharmaceuticals. The use of such compositions with multiple activeingredients is left to the discretion of the clinician.

A further embodiment of the present invention relates to a method forinducing a CTL response in a subject comprising administering tosubjects that express HLA-A1, -A2 or -A3 supertype antigens an effective(i.e., CTL-stimulating) amount of an immunogen described herein, e.g.,an amount sufficient to induce a CTL response to tumor cells expressingat least HLA-A1, HLA-A2, or HLA-A3, as the case may be, therebyeliciting a cellular response against said tumor cells.

A still further embodiment of the present invention relates to a methodfor inducing a CTL response in a subject, wherein the immunogen is inthe form of a polynucleotide. In one non-limiting example, the methodincludes administering to subjects that express HLA-A1, HLA-A2, orHLA-A3 at least one CTL epitope, wherein said epitope or epitopes areselected from a group comprising the peptides described herein, and arecoded within a polynucleotide sequence that does not include the entireprotein coding region, in an amount sufficient to induce a CTL responseto tumor cells expressing HLA-A1, HLA-A2, or HLA-A3.

Antibodies

The immunogens described herein can be used to stimulate the productionof antibodies for use in passive immunotherapy, for use as diagnosticreagents, and for use as reagents in other processes such as affinitychromatography.

The present invention also relates to antibodies that react withimmunogens, such as a polypeptide comprising one or more of the epitopicpeptides of SEQ ID NOs:1-21 (or a variant thereof) as described herein.Active fragments of such antibodies are also specifically contemplated.Such antibodies, and active fragments of such antibodies, for example,and Fab structure, can react with, including where it is highlyselective or specific for, an immunogenic structure comprising 2, 3, 4or more of the epitopic peptides described herein.

With the advent of methods of molecular biology and recombinanttechnology, it is now possible for the artisan of ordinary skill toproduce antibody molecules by recombinant means and thereby generategene sequences that code for specific amino acid sequences found in thepolypeptide structure of the antibodies. Such antibodies can be producedby either cloning the gene sequences encoding the polypeptide chains ofsaid antibodies or by direct synthesis of said polypeptide chains, within vitro assembly of the synthesized chains to form active tetrameric(H₂L₂) structures with affinity for specific epitopes and antigenicdeterminants. This has permitted the ready production of antibodieshaving sequences characteristic of neutralizing antibodies fromdifferent species and sources.

Regardless of the source of the antibodies or nanobodies, or how theartisan of ordinary skill chooses to produce such antibodies ornanobodies, including recombinantly constructed or synthesized, in vitroor in vivo, by using transgenic animals, such as cows, goats and sheep,or by using cell cultures in bioreactors, or by direct chemicalsynthesis employing no living organisms at any stage of the process, allantibodies and nanobodies have regions capable of interacting with astructurally complementary antigenic target. The regions interactingwith the target are referred to as “variable” or “V” regions and arecharacterized by differences in amino acid sequence from antibodies ofdifferent antigenic specificity.

The antibodies can also be wholly synthetic, wherein the polypeptidechains of the antibodies are synthesized and, possibly, optimized forbinding to the polypeptides disclosed herein as being receptors. Suchantibodies can be chimeric or humanized antibodies and can be fullytetrameric in structure, or can be dimeric and include only a singleheavy and a single light chain. Such antibodies can also includefragments, such as Fab and F(ab′)₂ fragments, capable of reacting withand binding to any of the polypeptides disclosed herein as beingreceptors.

Superagonist Peptides

The peptides and immunogens disclosed herein can also include internalmutations that render them “superantigens” or “superagonists” for T cellstimulation. Superantigen peptides can be generated by screening T cellswith a positional scanning synthetic peptide combinatorial library(PS-CSL) as described in Pinilla et al., 1992, Biotechniques, 13:901-5;Borras et al., 2002, J. Immunol. Methods, 267:79-97; US 2004/0072246;and Lustgarten et al., 2006, J. Immun., 176:1796-1805. When a native Tcell epitope is known, approximately 25% of the identified variants arefound to be superagonists. These can be up to 3 orders of magnitude moreeffective than the native ligand (Hemmer et al., 2000, J. Immunol., 164:861-871; La Rosa et al., 2001, Blood, 97:1776-86).

Positional scanning synthetic combinatorial libraries (PS-SCLs)representing trillions of peptides of different lengths can be used asunbiased sources of peptide antigens in T cell activation assays for theidentification of T cell epitopes. PS-SCL (Pinilla et al., 1992,Biotechniques, 13:901-905) are composed of systematically arrangedmixtures. In the case of a single position defined PS-SCL, each compoundpresent in a given mixture has a common individual amino acid at a givenposition, while the remaining positions are composed of mixtures of all19 natural L-amino acids (cysteine omitted). The screening data of agiven PS-SCL permits the identification of key residues at each positionof the peptide. It is important to note, however, that the activityfound for a mixture is due to the presence of specific active peptide(s)within the mixture, and not to the individual amino acids as separateentities. The combination of all amino acids defined in the most activemixtures leads to the active individual compounds.

Monitoring

The antigen-specific cellular immune responses of vaccinated subjectscan be monitored by a number of different assays, such as tetramerassays, ELISPOT, and quantitative PCR. The following sections provideexamples of protocols for detecting responses with these techniques.Additional methods and protocols are available. See e.g., CurrentProtocols in Immunology, Coligan, J. et al., Eds., (John Wiley & Sons,Inc.; New York, N.Y.).

Tetramers comprised of recombinant MHC molecules complexed with peptidecan be used to identify populations of antigen-specific T cells. Todetect T cells specific for antigens such as CD133, fluorochrome labeledspecific peptide tetramer complexes (e.g., phycoerythrin (PE)-tHLA)containing peptides from these antigens are synthesized and provided byBeckman Coulter (San Diego, Calif.). Specific CTL clone CD8 cells areresuspended at 10⁵ cells/50 μl FACS buffer (phosphate buffer plus 1%inactivated FCS buffer). Cells are incubated with 1 μl tHLA for 30minutes at room temperature and incubation is continued for 30 minutesat 4° C. with 10 μl anti-CD8 mAb (Becton Dickinson, San Jose, Calif.).Cells are washed twice in 2 ml cold FACS buffer before analysis byfluorescence-activated cell sorting (FACS) (Becton Dickinson).

ELISPOT assays can be used to detect cytokine secreting cells, e.g., todetermine whether cells in a vaccinated patient secrete cytokine inresponse to antigen, thereby demonstrating whether antigen-specificresponses have been elicited. ELISPOT assay kits are supplied from R & DSystems (Minneapolis, Minn.) and performed as described by themanufacturer's instructions. Responder (R) 1×10⁵ patients' PBMC cellsfrom before and after vaccination are plated in 96-well plates withnitrocellulose membrane inserts coated with capture Ab. Stimulator (S)cells (TAP-deficient T2 cells pulsed with antigen) are added at the R:Sratio of 1:1. After a 24-hour incubation, cells are removed by washingthe plates 4 times. The detection Ab is added to each well. The platesare incubated at 4° C. overnight and the washing steps will be repeated.After a 2-hour incubation with streptavidin-AP, the plates are washed.Aliquots (100 μl) of BCIP/NBT chromogen are added to each well todevelop the spots. The reaction is stopped after 60 min by washing withwater. The spots are scanned and counted with computer-assisted imageanalysis (Cellular Technology Ltd, Cleveland, Ohio). When experimentalvalues are significantly different from the mean number of spots againstnon-pulsed T2 cells (background values), as determined by a two-tailedWilcoxon rank sum test, the background values are subtracted from theexperimental values.

Quantitative PCR is another means for evaluating immune responses. Toexamine IFN-γ production in patients by quantitative PCR, cryopreservedPBMCs from patients' pre-vaccination and post-vaccinations samples andautologous dendritic cells are thawed in RPMI DC culture medium with 10%patient serum, washed and counted. PBMC are plated at 3×10⁶ PBMCs in 2ml of medium in 24-well plate; dendritic cells are plated at 1×10⁶/mland are pulsed 24 hour with 10 μg/ml tumor peptide in 2 ml in each wellin 24 well plate. Dendritic cells are collected, washed, and counted,and diluted to 1×10⁶ /ml, and 3×10⁵ (i.e., 300 μl solution) added towells with PBMC (DC: PBMC=1:10). 2.3 μl IL-2 (300 IU/mL) is added every3-4 days, and the cells are harvested between day 10 and day 13 afterinitiation of the culture. The harvested cells are then stimulated withtumor cells or autologous PBMC pulsed with 10 μg/ml tumor peptide for 4hours at 37° C. On days 11-13, cultures are harvested, washed twice,then divided into four different wells, two wells using for control(without target); and another two wells CTL co-cultured with tumor cells(1:1) if tumor cells are available. If tumor cells are not available, 10μg/ml tumor lysate is added to CTL. After 4 hours of stimulation, thecells are collected, RNA extracted, and IFN-γ and CD8 mRNA expressionevaluated with a thermocycler/fluorescence camera system. PCRamplification efficiency follows natural log progression, with linearregression analyses demonstrating correlation coefficients in excess of0.99. Based on empirical analysis, a one-cycle difference is interpretedto be a two-fold difference in mRNA quantity, and CD8-normalized IFN-γquantities are determined. An increase of >1.5-fold in post-vaccinerelative to pre-vaccine IFN-γ is the established standard for positivetype I vaccine responsiveness.

Ex Vivo Methods

The following protocol can be used to produce antigen-presenting cellsand/or antigen-specific CTL in vitro from patient-derived PBMC. Togenerate dendritic cells, the plastic adherent cells from PBMCs arecultured in AIM-V medium supplemented with recombinant human GM-CSF andrecombinant human IL-4 at 37° C. in a humidified CO₂ (5%) incubator. Sixdays later, the immature dendritic cells in the cultures are stimulatedwith recombinant human TNF-α for maturation. Mature dendritic cells arethen harvested on day 8, resuspended in PBS at 1×10⁶ per mL with peptide(2 μg/mL), and incubated for 2 hours at 37° C.

Autologous CD8+ T cells are enriched from PBMCs using magneticmicrobeads (Miltenyi Biotech, Auburn, Calif.). CD8+ T cells (2×10⁶ perwell) are co-cultured with 2×10⁵ per well peptide-pulsed dendritic cellsin 2 mL/well of AIM-V medium supplemented with 5% human AB serum and 10units/mL rhIL-7 (Cell Sciences) in each well of 24-well tissue cultureplates. About 20 U/ml of IL-2 is added 24 h later at regular intervals,2 days after each restimulation. On day 7, lymphocytes are restimulatedwith autologous dendritic cells pulsed with peptide in AIM-V mediumsupplemented with 5% human AB serum, rhIL-2, and rhIL-7 (10 units/mLeach). About 20 U/ml of IL-2 is added 24 h later at regular intervals, 2days after each restimulation. On the seventh day, after the threerounds of restimulation, cells are harvested and tested the activity ofCTL. The stimulated CD8+ cultured cells (CTL) are co-cultured with T2cells (a human TAP-deficient cell line) pulsed with 2 μg/ml CD133peptides. After 24 hours incubation, IFN-γ in the medium is measured byELISA assay.

Animal Models

Vaccination (e.g., DC vaccination) can be evaluated in animal models.Suitable models for cancers include injection models, in which cells ofa tumor cell line are injected into the animal, and genetic models, inwhich tumors arise during development. In some cases, a transgenicanimal (e.g., a mouse) that expresses an HLA (e.g., HLA-A2) can be used.See, e.g., Choi et al., 2002, J. Immunol. Methods, 268:35-41.

To evaluate dendritic cell vaccination in an animal model, functionaldendritic cells are isolated from bone marrow derived cells of theanimal and differentiated in vitro in the presence of cytokines, asdetailed above. Mature dendritic cells are pulsed with tumor antigens(e.g., tumor antigens derived from the tumor cell line that will beimplanted into the animal or synthetic peptides corresponding toepitopes of those antigens). Animals are implanted with cells of thetumor cell line. After implantation, animals are vaccinated withantigen-pulsed dendritic cells one or more times. Survival and immuneresponsiveness is measured.

Kits

The present invention is also directed to kits to treat cancers. Thekits are useful for practicing the methods described herein for treatingcancer with a vaccine comprising an antigen or APCs loaded with anantigen as described herein. The kit is an assemblage of materials orcomponents, including at least one of the compositions described herein.Thus, in some embodiments, the kit includes a set of peptides for use invaccination or preparing cells for vaccination. The kit can also includeagents for preparing cells (e.g., cytokines for inducing differentiationof DC in vitro). The invention also provides kits containing acomposition including a vaccine comprising dendritic cells (e.g.,cryopreserved dendritic cells) loaded with the antigens as describedherein.

The exact nature of the components configured in the kit depends on itsintended purpose. For example, some embodiments are configured for thepurpose of treating brain cancer, colon cancer, hepatocellularcarcinoma, prostate cancer, multiple myeloma, and melanoma. In oneembodiment the brain cancer is a glioma. In another embodiment, thebrain cancer is glioblastoma multiforme (GBM). In another embodiment,the brain cancer is an astrocytoma. In one embodiment, the kit isconfigured particularly for the purpose of treating mammalian subjects.In another embodiment, the kit is configured particularly for thepurpose of treating human subjects. In further embodiments, the kit isconfigured for veterinary applications, treating subjects such as, butnot limited to, farm animals, domestic animals, and laboratory animals.

Instructions for use can be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to treat cancer. For example, the instructions can includeinstructions to administer a vaccine (e.g., comprising dendritic cellsloaded with the antigens described herein) to the patient. Instructionsfor use can also include instructions for repeated administrations ofthe vaccine; for example, administering the three doses of the vaccinein two week intervals.

Optionally, the kit also contains other useful components, such as,diluents, buffers, pharmaceutically acceptable carriers, syringes,catheters, applicators, pipetting or measuring tools, or other usefulparaphernalia as will be readily recognized by those of skill in theart.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as compositions describedherein and the like. The packaging material is constructed by well knownmethods, preferably to provide a sterile, contaminant-free environment.The packaging materials employed in the kit are those customarilyutilized in cancer treatments or in vaccinations. As used herein, theterm “package” refers to a suitable solid matrix or material such asglass, plastic, paper, foil, and the like, capable of holding theindividual kit components. Thus, for example, a package can be a glassvial used to contain suitable quantities of a composition containing avaccine, e.g., a vaccine comprising an immunogen or dendritic cellsloaded with the antigens as described herein. The packaging materialgenerally has an external label which indicates the contents and/orpurpose of the kit and/or its components.

EXAMPLES

The following Examples are illustrative and not limiting.

Example 1 Prediction and Synthesis of CD133 Epitopes

Epitopes of CD133 were predicted using the Immune Epitope free publicdatabase for predicting Class 1 MHC-peptide binding to HLA-A*0101,-A*0201 and -A*0301 using the artificial neural network (ANN) method(Nielsen et al., 2003, Protein Sci., 12:1007-17). The algorithm used befound on the World Wide Web attools.immuneepitope.org/analyze/html/mhc_binding.html. Peptides wereselected based on predicted IC₅₀ for HLA-A*0101 (IC50≦5000 nM),HLA-A*0201 (IC50≦500 nM), and HLA-A*0301 (IC50≦500 nM). Eighty-fourcandidate nine-amino acid sequences of CD133 were synthesized (ProImmuneLtd., Oxford, UK) to determine whether the peptides bound to the MHCsHLA-A*0101, HLA-A*0201, and HLA-A*0301. The information generated fromthe Immune Epitope Database is only a general guideline. These scoresare based solely on algorithms and cannot confirm whether the sequencesare true or optimal T cell epitopes. Additionally, there may be somesequences that do not score well on algorithms that will be good T cellepitopes.

Example 2 Identification of HLA-A*0101 Epitopes

Eighteen of the candidate peptides were assembled with HLA-A*0101 andanalyzed for MHC binding to determine their level of incorporation intoMHC molecules using the REVEAL™ MHC binding assay (ProImmune Ltd.,Oxford, UK). Binding to MHC molecules was compared to that of two knownT-cell epitopes: an intermediate control peptide and a positive controlpeptide with weak and very strong binding properties, respectively.Three peptides bound to HLA-A*0101 at at least 50% of the level of thepositive control, as indicated on Table 1.

TABLE 1 HLA-A*0101 Binding SEQ ID NO Sequence % Positive Control 1AVDVFLCSY 58.58 2 SSELESLKV 74.14 3 IIDPLNLFW 91.46 Intermediate —   2.25 ± 1.2 Control Positive —  100.00 ± 15.2 Control

The peptides that bound to HLA-A*0101 were synthesized as ProVE®pentamers (ProImmune Ltd., Oxford, UK) for further analysis. Thepentamers were subjected to incubation and analysis at 37° C. todetermine the stability of peptide-MHC complexes. Off-rates of thecomplexes were measured by REVEAL™ off rate analysis (ProImmune Ltd.,Oxford, UK) after 0, 2,and 24 hours of incubation at 37° C. Theoff-rates of the peptides in terms of t_(1/2) half-life values arepresented in Table 2.

TABLE 2 HLA-A*0101 Off Rates SEQ ID NO Sequence t_(1/2) (h) 1 AVDVFLCSY20.71 2 SSELESLKV 0.46 3 IIDPLNLFW 0.35 Intermediate — 0.29 ControlPositive — 102.74* Control *: The measurement interval of 24 hours wastoo short to calculate this value accurately.

Example 3 Identification of HLA-A*0301 Epitopes

Fifteen of the candidate peptides were assembled with HLA-A*0301 andanalyzed for MHC binding to determine their level of incorporation intoMHC molecules using the REVEAL™ MHC binding assay (ProImmune Ltd.,Oxford, UK). Binding to MHC molecules was compared to that of two knownT-cell epitopes: an intermediate control peptide and a positive controlpeptide with weak and very strong binding properties, respectively. Sixpeptides bound to HLA-A*0301 at at least 45% of the level of thepositive control, as indicated on Table 3.

TABLE 3 HLA-A*0301 Binding SEQ ID NO Sequence % Positive Control 4KLFNKSKMK 80.81 5 ILAQYNTTK 46.61 6 YLSGKLFNK 46.87 7 RTRIKRSRK 87.33 8LSSSLTSVK 52.49 9 NLFWFGIGK 53.08 Intermediate —     9.19 ± 1.5 ControlPositive —   100.00 ± 4.6 Control

Five of the peptides that bound to HLA-A*0301 were synthesized as ProVE®pentamers (ProImmune Ltd., Oxford, UK) for further analysis. Thepentamers were subjected to incubation and analysis at 37° C. todetermine the stability of peptide-MHC complexes. Off-rates of thecomplexes were measured by REVEAL™ off rate analysis (ProImmune Ltd.,Oxford, UK) after 0, 2,and 24 hours of incubation at 37° C. Theoff-rates of the peptides in terms of t_(1/2) half-life values arepresented in Table 4.

TABLE 4 HLA-A*0301 Off Rates SEQ ID NO Sequence t_(1/2) (h) 4 KLFNKSKMK27.81* 5 ILAQYNTTK 2.07 7 RTRIKRSRK 23.92 8 LSSSLTSVK 1.80 9 NLFWFGIGK3.35 Intermediate — 0.56 Control Positive — 49.82* Control *: Themeasurement interval of 24 hours was too short to calculate these valuesaccurately.

Example 4 Identification of HLA-A*0201 Epitopes

Fifty-five of the candidate peptides were assembled with HLA-A*0201 andanalyzed for MHC binding to determine their level of incorporation intoMHC molecules using the REVEAL™ MHC binding assay (ProImmune Ltd.,Oxford, UK). Binding to MHC molecules was compared to that of two knownT-cell epitopes: an intermediate control peptide and a positive controlpeptide with weak and very strong binding properties, respectively.Eleven peptides bound to HLA-A*0201 as well or better than the positivecontrol, as indicated on Table 5.

TABLE 5 HLA-A*0201 Binding SEQ ID NO Sequence % Positive Control 10VLDEIKSMA 309.26 11 YLQWIEFSI 112.67 12 NLLSFAYDL 179.71 13 FITNNTSSV107.15 14 RVLDTPYLL 165.18 15 SLDFAQNFI 114.58 16 ELVHIFLYV 155.93 17LVLGSLLLL 101.90 18 SQLNSNPEL 123.10 19 ILCCVLGLL 163.06 20 GLLERVTRI131.30 21 FLLPALIFA 93.59 Intermediate —     2.65 ± 2.1 Control Positive—   100.00 ± 6.7 Control

Twelve of the peptides that bound to HLA-A*0201 were synthesized asProVE® pentamers (ProImmune Ltd., Oxford, UK) for further analysis. Thepentamers were subjected to incubation and analysis at 37° C. todetermine the stability of peptide-MHC complexes. Off-rates of thecomplexes were measured by REVEAL™ off rate analysis (ProImmune Ltd.,Oxford, UK) after 0, 2,and 24 hours of incubation at 37° C. Theoff-rates of the peptides in terms of t_(1/2) half-life values arepresented in Table 6.

TABLE 6  HLA-A*0201 Off Rates SEQ ID NO Sequence t_(1/2) (h) 10VLDEIKSMA 1.43 11 YLQWIEFSI 83.00* 12 NLLSFAYDL 1.83 13 FITNNTSSV 19.9814 RVLDTPYLL 3.51 15 SLDFAQNFI 15.49 16 ELVHIFLYV 6.86 17 LVLGSLLLL 8.2518 SQLNSNPEL 2.37 19 ILCCVLGLL 5.63 20 GLLERVTRI 74.30* 21FLLPALIFA >120* Intermediate - 16.18 Control Positive - 55.67* Control*: The measurement interval of 24 hours was too short to calculate thesevalues accurately.

Example 5 Generation of Superagonist CD133 Peptides

Superagonist peptides of the CD133 epitopes described herein areproduced by the methods described below. These peptide superagonistsexhibit a superior capacity to induce CTL responses.

For this application T cell lines and clones are generated fromperipheral blood mononuclear cells (PBMC) derived from glioma patients.Epstein Barr transformed autologous B cells are used as antigenpresenting cells through all the T cell functional assays andstimulations. Blood is obtained from glioma patients and carefullylayered on top of 50 ml conical tubes (polypropylene, Sarsted) in aratio of 2 volumes per 1 volume of Histopaque (Sigma, St Louis, Mo.).Each tube is then placed in a clinical swing out centrifuge (Beckman)and spun down for 30 minutes at 400 g at room temperature. The PBMC arethen collected from the interface with a transfer plastic pipette(Samco) and washed 2× with D-PBS at 250 g and 1× with culture medium(IMDM, Bio-whittaker, Walkersville, Md.) containing 8% AB human serum(Gemini Bio-products, Woodland, Calif.) at 200 g for 10 minutes eachstep. The supernatant is aspirated and discarded, and the cells areresuspended in culture medium.

CD8+ and CD4+ T cells are isolated from PBMC by positive selectionfollowing manufacturer's instructions (CD8 and CD4 positive selectionkits, Dynal Biotech Inc., Lake Success, N.Y.). The isolated cells areused immediately for stimulation protocols.

Transformation of B cells from PBMC by Epstein Barr virus (EBV) isperformed immediately after PBMC isolation. Briefly, frozen PBMCs arethawed, washed, and resuspended in CRPMI 10% FBS. 5 to 10 million PBMCsare resuspended in 2.5 ml of CRPMI 10% FBS. Then, 2.5 ml of thawedsupernatant from B95.8 Marmoset cells (containing the EBV) are added toeach conical tube containing the cells. The cells are incubated for 2hours in a water bath at 37° C. CRPMI 10% FBS containing 1 μg/ml ofCyclosporin A is then added to each tube. 10 ml suspensions aretransferred to T-25 flasks and incubated for 3 weeks. At this point, thecells form clumps visible to the naked eye. By microscopic examination,the cells appear large, clear and possibly hairy. These are indicatorsof B cell immortalization by EBV. Cells are mixed in their flasks andthe 10 ml culture is split into 2 new T-25 flasks (5 ml each). 5 ml offresh CRPMI-10 media containing 1 μg/ml cyclosporin A is added to eachflask and the cultures are incubated for 1 week at 37° C. At this timepoint, an aliquot of each donor's cells is stained for CD19 expression(Pharmingen anti-CD19-APC stain) and analyzed by flow cytometry. Thecell lines are then expanded and frozen down at 5×10⁶/vial. ImmortalizedB cells are expanded in culture by splitting 1:3 in CRPMI-10 media(without cyclosporin A) in T-25 flasks once a week and incubating at 37°C., 5% CO₂. These lymphoblastoid B cell lines (EBV-LCL) are used asantigen presenting cells in the following T cell functional assays.

PBMC are stimulated with the reported CD133 antigens and with cancerstem cell lines in the presence of autologous dendritic cells. Briefly,T cells derived from either single well or multiple wells (bulkcultures) are used after 6-7 days of stimulation. T cell limitingdilutions are done at a concentration of 0.3, 1, 3 and 10 cells/well in96-well round bottom plates (Corning). 1×10⁵ irradiated autologousdendritic cells per well are added together with IL-2 and IL-7 (20 U/mland 10 ng/ml, respectively). About five to ten times the original numberof the plated cells is obtained. Wells that demonstrate growth areexpanded by restimulation with a larger number of irradiated allogeneicfeeders, phytohemagglutinin (PHA), and IL-2 until sufficient numbers areobtained for specificity tests. At this point, some cells are frozenwhile others are tested for antigen reactivity by using differentreadouts of T cell activation, namely cytokine production, cell killingand proliferation. Multiplex cytokine assay (Millipore, Billerica,Mass.) is performed according to the manufacturer's instructions toquantify, in an unbiased manner, a large cytokine spectrum to determinethe best cytokine(s) for the evaluation of antigen specificity.

TCR profiles of the generated T cell clones are obtained to demonstrateand monitor clonality. The Vβ repertoire is determined using flowcytometry (as described above) with specific mAbs (available throughImmunotech, Miami, Fla.) for cells that expand to large numbers (>10million).

Immortalization of the antigen-responsive human T cells from PBMCsprovides an advantage for the study of their fine specificity with thecombinatorial libraries, because a high number of T cells are needed forthe screening of the these libraries. Indeed, in order to obtainadequate data from combinatorial libraries, cells should be grown to aminimal of 30 to 100 million cells. For this reason, we the defined Tcell lines and clones will be immortalized. Briefly, transduction isobtained by magnetofection in dividing T cells (recently stimulated),which are washed, counted, and plated with 100 U/ml of IL-2 in completemedium in 96 well plates (flat bottom). A mixture of the retroviralvector with Viromag R/L (OZ Biosciences) is incubated for 20 minutesbefore being layered onto the T cells, and the plate is then carefullyset on the top of magnetic plate and incubated overnight. The next daythe cells are resuspended in fresh complete medium with IL-2 andtransferred to a larger well. After 48 hours the transfection efficiencyis assessed by flow cytometry by staining with anti-NGFR-PE. Magneticbead enrichment of transduced cells is performed according to Miltenyiprotocols using anti-PE beads (Miltenyi).

CD133-specific T cell lines and clones are obtained within 2-4 monthsfrom the primary stimulation.

Combinatorial peptide libraries for screening for superagonist peptidesare prepared as described previously in Pinilla et al., 1994, Biochem.J., 301:847-853.

T cell functional assays are performed in 96-well plates (Corning Inc.,Corning, N.Y.). Each plate can accommodate 80 samples in columns 3-12,with the first 2 columns reserved for negative and positive controlwells. The dispensing of samples and common reagents is accomplishedusing a Precision Biotek automated liquid handling instrument (Biotek,Winooski, Vt.). All samples, both libraries and individual compounds,are stored in 96-tube racks that are compatible with both the 96-wellplates and the liquid handler instrumentation. Thirty plates per weekare tested with the T cell functional assays. For assays that are run induplicate, this generates approximately 1,000 data points per week. Dataare acquired in the instruments specified for each type of assay andtransferred to specifically designed Excel workbooks for rapid andaccurate analysis.

Library mixtures are tested at a final concentration of 100 or 200 μg/mlusing the general plate layout described above. Briefly, 25,000 T cellsare cultured in the presence of 50,000 irradiated autologouslymphoblastoid cell lines (LCLs) and 25 μl of each mixture library at 2mg/ml in complete RPMI. Each mixture is tested in duplicate. Controlwells include T cells and LCLs without mixtures and with or without PHA(at a final concentration of 5 ug/ml). As mentioned before for antigenspecificity, different readouts of T cell activation are tested toconfirm the assay readout that provides the best signal for thescreening with the libraries. After the screening with the library, theresults are used to design individual peptides by combining theselection of the defined amino acids of the most active mixtures at eachdefined position. This approach provides optimized agonists andsuperagonist peptides of the CD133 epitopes described herein. The mostactive peptides are selected to determine their in vitro immunogenicityand cross reactivity with the native antigen.

Individual agonist and superagonist peptides are synthesized by thesimultaneous multiple peptide synthesis method (Houghten, 1985, Proc.Natl. Acad. Sci. USA, 82:5131-35). The purity and identity of eachpeptide are characterized using an electrospray mass spectrometerinterfaced with a liquid chromatography system.

To test the stimulatory capacity of the peptides, 25,000 T cells arecultured in the presence of 50,000 irradiated autologous LCLs and eachof the individual peptides at a final concentration of 10 and 1 μg/ml.The stimulatory activity of the positive peptides is determined withdose-titration experiments to determine the concentration that yields50% stimulatory activity (EC-50).

These studies identify superagonist peptides derived from the CD133described herein. Strong agonist peptides recognized with EC-50 valuesin the nanomolar range are identified.

Example 6 Immunization with CD133 Peptides

Vaccination with CD133 epitope peptides described herein andsuperagonists thereof is tested for killing of tumors in humanizedHLA-A2 transgenic mice. Similar methods can also be performed in micetransgenic for other HLA (see, e.g., Alexander et al., 1997, J.Immunol., 159:4753-61). The efficacy of vaccination with CD133 epitopeand its superagonists with regard to peripheral cytotoxicity,intracranial tumor infiltration, and survival is tested.

Briefly, HHD mice are immunized with an epitope peptide described hereinemulsified in Incomplete Freund's adjuvant and helper antigen. Bulkpopulations of splenocytes are tested for specific cytotoxicity againstthe EL4-HHD cells pulsed with the peptide, control unpulsed EL4-HHD, orEL4-HHD- peptide cells. Measurement of the peptide/HLA complex bindingand stability is performed. Survival of animals vaccinated with CD133epitope superagonists is compared.

CD133 peptides and superagonists are synthesized byN-(9-fluorenyl)methoxycarbonyl chemistry at >95% purity as indicated byanalytic high-performance liquid chromatography and mass spectrometricanalysis. Peptides are dissolved in PBS/10% DMSO at a concentration of 2mg/ml and stored at −20° C. until use.

The peptides are tested in HHD mice, which are humanized with regard toHLA-A2 expression (Pascolo et al., 1997, J. Exp. Med., 185:2043-51). TheHHD mice used are Dbcβ2 microglobulin null and transgenic for modifiedHLA-A*0201-β2 microglobulin single chain (HHD) (Eguchi et al., 2006,Cancer Res., 66:5883-91; Gross et al., 2004, J. Clin. Invest.,113:425-433).

An HHD-syngeneic tumor cell line that expresses CD133 is created. Thefull-length human CD133 cDNA fragment is generated by reversetranscription-PCR using forward (AGTATGGCTTTCGTTTGCTTGGC; SEQ ID NO:22)and reverse (TACCGAGCTCGGATCCACTAGT; SEQ ID NO:23) primers and CSC1glioblastoma multiforme cancer stem cell-derived total RNA. The CD133cDNA is then cloned into the expression plasmid pEF6/V5-His-TOPO vector(Invitrogen) to generate pEF6/V 5-CD133. EL4-HHD cells are thentransfected with the pEF6/V5-CD133 using Cell Line Nucleofector kit T(Amaxa, Gaithersburg, Md.), and a blasticidine-resistant clone thatstably expresses the highest level of CD133 based on flow-cytometryusing CD133 mAb (Tessa) is selected (EL4-HHD-CD133) for further use.

Cells are stained with phycoerythrin-conjugated HLA-A*0201/peptidetetramers (10 μg/mL) in PBS containing 1% bovine serum albumin for 15minutes at room temperature, washed once, and stained withFITC-conjugated anti-human CD8 or anti-mouse CD8 (BD Biosciences, SanDiego, Calif.). Flow cytometric analyses are performed using CoulterEPICS cytometer (Beckman Coulter, Fullerton, Calif.).

To measure the peptide/HLA-A2 complex binding and stability, T2 cells(1×10⁶ cells/mL) are incubated with various concentrations (0.1-100nmol/L) of peptides in serum-free RPMI 1640 at 37° C. overnight in anatmosphere containing 5% CO₂. The cells are then washed twice with PBSand stained with the BB7.2 mAb for 30 minutes at 4° C. After washing,FITC-conjugated goat anti-mouse IgG (Caltag, Burlingame, Calif.) is usedas the secondary antibody. Surface expression levels of HLA-A2 areexamined by flow cytometry. Peptide binding is evaluated by determiningmean fluorescence intensity (MFI).

HHD mice are vaccinated (on days 0 and 7) with s.c. injections of 100 μgof peptide or superagonist emulsified in Incomplete Freund's adjuvant(IFA; Difco, Detroit, Mich.) in the presence of 140 μg of theI-Ab-restricted HBVcore128 T-helper epitope, which stimulates a CD4+helper T-cell response. Control animals receive IFA containing HBVhelper-peptide only. On day 11 after the second immunization, theanimals are sacrificed, and 5×10⁷ splenocytes are stimulated in vitrowith the same peptide that is used for in vivo stimulation (10 μmol/L).On day 6 of culture, the bulk populations are tested for specificcytotoxicity against EL4-HHD or EL4-HHD-peptide cells.

To assess systemic protective immunity against i.c. tumor challenge, onday 7 after the second immunization, HHD mice receive an i.c.inoculation of EL4-HHD-peptide cells. Briefly, 5×104 EL4-HHD-peptidecells are stereotactically injected through an entry site at the bregma2 mm to the right of the sagittal suture and 3 mm below the surface ofthe skull of anesthetized mice using a stereotactic frame. The animalsare monitored daily after treatment for the manifestation of anypathologic signs.

Mice bearing i.c. EL4-HHD-peptide tumors receive immunizations on days14 and 21 after the tumor inoculation, sacrificed by CO₂ asphyxiation onday 28, and perfused through the left cardiac ventricle with PBS. Brainsare enzymatically digested (Walker et al., 2000, J. Immunol., 1653128-35; Calzascia et al., 2005, Immunity, 22:175-184), and cells fromeach brain are resuspended in 70% Percoll (Sigma, Saint Louis, Mo.),overlaid with 37% and 30% Percoll and centrifuged for 20 minutes at500×g. Enriched brain-infiltrating lymphocyte (BIL) populations arerecovered at the 70% to 37% Percoll interface.

Survival data are compared using a log-rank test. Comparative numbers ofT-cell responses are analyzed by Student's t test for two samples withunequal variances. Statistical significance is determined at the <0.05level. Positive response is also defined as follows: the specific lysisby the responder cells against antigen-positive target cells is at least15% and 2-fold higher than lytic levels by corresponding controlconditions in at least two effector/target (E/T) ratios. Post-hoccontrasts (e.g., Student's ‘t’ test) are performed to determinesignificant differences, i.e., p<0.05 between the 3 groups of animalsreceiving epitope vaccination, control vaccinations, and PBS vehiclecontrol. 10 animals/group are used, sufficient to detect a 1.2 SDdifference between groups at a power of 0.8 and a p=0.05.

Brain inflammation in response to vaccination is measured by performinga quantitative stereological analysis of the infiltration of T, B, andNK lymphocytes and macrophages. An immune cellular infiltrate isdetected only in the intracranial tumor. Influx of CD4+, CD8+, and NKcells is observed within the tumor and peritumoral area. Increasedactivation of astrocytes is also observed, as evidenced by up-regulationof GFAP immunoreactivity in astrocytes.

Example 7 Induction of Immune Responses Using CD133 SuperagonistPeptides

The capacity of CD133 superagonist peptides to induce CTLs capable ofcross-reacting against the wild-type epitope is determined. HLA bindingand stability assays are performed to determine whether the improvedimmunogenicity of the analog peptides is at least partially attributableto higher binding/stability of these superagonist peptides in HLAcomplexes that are required for specific CTL recognition. CTL assaysanalyzing reactivity versus peptide dose titration on T2 target cellsare performed to detect whether the CTLs developed using thesuperagonist peptides possesses a higher functional avidity than thoseprimed with wild-type peptide. CTL clones raised against the agonisticpeptide-epitope have a more restricted T cell receptor (TCR) usage andhigher TCR functional avidity than the CTL clones raised against thenatural peptide-epitope.

PBMCs are obtained from glioma patients and healthy donors under anInstitutional Review Board-approved protocol. HLA (e.g., HLA-A2)expression on the PBMC is validated using the monoclonal antibodies(mAb) MA2.1 (against HLA A2, B17) and BB7.2 (against HLA A2, Aw69: bothfrom the American Type Culture Collection, Manassas, Va.) in indirectimmunofluorescence assays monitored by flow cytometry.

HLA-A*0201 restricted CTL clones specific for CD133 natural andsuperagonist peptide-epitopes are generated in vitro. Harvested maturemonocyte derived dendritic cells (mMoDC) are pulsed with natural andsuperagonist peptides (20 μM) and, after washing, are mixed withmagnetically enriched CD8+ T cells from either thawed cryopreserved CD14negative PBMCs or fresh PBMCs. Peptide pulsed mMoDC and enriched CD8+ Tcells are mixed at a ratio of 1:20 in the presence of sCD40L (2 μg/ml)to initiate Th1-polarization of mMoDC, which boosts IL-12 production(Mailliard et al., 2002, J. Exp. Med., 195:473-483; Mailliard et al.,2004, Cancer Res., 64: 5934-37). On day three, the priming culture issupplemented with IL-2 (50 U/ml) and IL-7 (10 U/ml), and on day 12 theculture is restimulated with peptide pulsed autologous PBMC. At day24-28, the priming culture is tested by tetramer staining for thepresence of expanded primed CTL specific for the peptide used. As apositive control, priming with HLA-A*0201 restricted p24HIV-1 (SLYNVATL;SEQ ID NO:24) is run concurrently (Kan-Mitchell et al., 2006, J.Immunol., 176:6690-6701; Mitchell et al., 2007, Cancer Immunol.Immunother., 56:287-301).

For assessment of stability, patient-derived T2 cells (1×10⁶ per mL) areincubated overnight with 100 μmol/L of each peptide in serum-free RPMI1640 at 37° C. Thereafter, the cells are washed four times to removefree peptides and incubated at 37° C. for 0, 3, or 6 hours. The cellsare stained with the BB7.2 mAb to evaluate the HLA-A2 moleculeexpression at each time point. Peptide-induced HLA-A2 expression isevaluated by calculating the mean fluorescence of peptide-incubated T2cells minus the mean fluorescence of T2 cells in the absence of peptide.DC50 is measured as the time required for the loss of 50% of theHLA-A2/peptide complexes stabilized at t=0.

TCR usage of CTL clones is determined by expression of variable regionof β chain (V-β) of TCR. Expression of TCR-V-β and V-α among clonallyexpanded CD8 T cells is assessed by a real-time PCR using a fluorogenicprobe (Lang et al., 1997, J. Immunol. Methods, 203:181-192). This methodoffers a similar degree of sensitivity to the conventional detection ofTCR-V-β expression with reduced processing time. Briefly, total RNAextraction and reverse transcription are performed. In the PCR step, aV-β-specific 5′ probe, common CB 3′ primers, and an internal fluorogenicprobe are used to amplify 26 possible V-β genes. The detection andquantitation of PCR products are done by using a 7900HT Fast Real-TimePCR System (Applied Biosystems), with which it is possible to calculatethe semi-quantitative ratio of TCR V-β expression among clonallyexpanded CD8 T cells. Once the expression of a particular TCR V-β isdetermined, using the same V-β specific primer the sequencecorresponding to CDR 3 is determined. This allows for delineation of theclonality of CTLs.

Tetramer decay analysis is performed to determine TCR avidity of the CTLclones (Savage et al., 1999, Immunity, 10:485-492). CTL clones arestained with tetramer (1-25 nM), as in the equilibrium bindingexperiments above. Cells are washed twice with FACS buffer (4% FCS and0.1% sodium azide in PBS) and kept on ice until they are mixed withexcess anti-HLA-A02 mAb (BB7.2, BD Bioscienes) and then incubated atroom temperature to allow for tetramer dissociation. The anti-HLA-A02mAb is used to block rebinding of tetramer to the TCR. Dissociation isfollowed for 0-180 minutes, after which cells are washed quickly withice-cold buffer to remove all unbound tetramer and blocking mAb. Thecells are then fixed for flow cytometry analysis (CyanADP,Beckman-Coulter). The natural logarithm of percentage of Geometric MeanFluorescence (GMF) at each time point (compared with 0 minutes) isplotted against time. The half-life of each pMHC multimer is derivedfrom the slope by the equation t1/2−1n2/slope.

CTL activity of the in vitro primed CTL clones is measured by flowcytometric assay (Betts et al., 2003, J. Immunol. Methods, 281:65-78;Betts et al., 2004, Methods Cell Biol., 75:497-512). Briefly, thepriming culture containing the CTL clone is mixed 1:1 with peptidepulsed T2 cells for 6 hours in the presence of CD107a, Monensin, andBrefeldin A. After the 6 hour incubation, cells are stained withcorresponding tetramers and anti-CD8 mAb, followed by intracellularIFN-γ and TNFα staining Stained cells are run on Beckman-Coulter CyanADP(9 color, 11 parameters) for flow cytometric analysis. All the assaysare run in triplicate.

To measure cytotoxicity, targets are labeled with 100 μCi of ⁵¹Cr for 60minutes, plated in 96-well V-bottomed plates (3×10³ cell/well), andpulsed with peptides (1 μM) at 37° C. for 2 hours. Effectors are addedand incubated at 37° C. for an additional 4 hours. One hundred μl ofsupernatant are collected, and the radioactivity is measured in a gammacounter. The percentage of specific lysis is determined as:(experimental release−spontaneous release)/(maximal release−SpontaneousRelease)×100.

As a surrogate marker for CTL responses, cytokine responses, such asIFN-γ (Mailliard et al., 2004, Cancer Res., 64:5934-37; Herr et al.,2000, Blood, 96:1857-64) and IL-2 (Carrabba et al., 2003, Cancer Res.,63: 1560-67) can be monitored. IFN-γ and IL-2 secretion levels from CTLcultures stimulated with native or superagonist peptides are measuredusing cytokine-specific ELISA and IFN-γ enzyme-linked immunospot assays.

The relative affinity (RA) of CD133 superagonist peptides for HLA-A*0201are measured. Briefly, T2 cells are incubated with variousconcentrations of peptides ranging from 100 to 0.1 μM overnight and thenstained with BB7.2 mAb to quantify the surface expression of HLA-A*0201allele. For each peptide concentration, the HLA-A*0201-specific stainingis calculated as the percentage of staining obtained with 100 μM of thereference peptide HIVpol589 (IVGAETFYV; SEQ ID NO:25). The RA isdetermined as: RA=(concentration of each peptide that induces 20% ofHLA-A*0201 expression/concentration of the reference peptide thatinduces 20% of HLA-A*0201 expression).

The stability of superagonist peptide/HLA-A*0201 complexes is assessed.Briefly, T2 cells are incubated overnight with 100 μM of each peptide.Cells are then incubated with Brefeldin A (Sigma, St. Louis, Mo.) at 10μg/ml for 1 hour, washed, incubated at 37° C. for 0, 2, 4, or 6 hours inthe presence of Brefeldin A (0.5 μg/ml), and then stained with BB7.2mAb. For each time point, peptide induced HLA-A*0201 expression iscalculated as: mean fluorescence of peptide preincubated T2 cells-meanfluorescence of T2 cells treated in similar conditions in the absence ofpeptide. DC50 is defined as the time required for the loss of 50% of theHLA-A*0201/peptide complexes stabilized at t=0.

CTL are generated from human PBMCs. PBMCs are collected by leukapheresisfrom healthy HLA-A*0201 volunteers. Dendritic cells are produced fromadherent cells (2×10⁶ cells/ml) cultured for 7 days in the presence of500 IU/ml granulocyte macrophage colony-stimulating factor (Leucomax;Schering-Plough, Kenilworth, N.J.) and 500 IU/ml IL-4 (R&D Systems,Minneapolis, Minn.) in complete medium [RPMI 1640 supplemented with 10%heat-inactivated human AB serum, 2 μM L-glutamine (Invitrogen) andantibiotics]. On day 7, dendritic cells are collected and pulsed with 40μg/ml peptide in the presence of 3 μg/ml β2 m (Sigma) for 4 hours at 20°C. and then irradiated (4200 rad). CD8+ T cells are isolated by positiveselection with immunomagnetic beads (Miltenyi Biotec, Bergisch Gladbach,Germany) according to the manufacturer's instructions. A total of0.5×10⁶ CD8+ T cells are cocultured with 0.25×10⁵ dendritic cells in afinal volume of 0.5 ml/well in a 48-well plate in the presence of 10ng/ml IL-7 (R&D Systems). Human IL-10 (R&D Systems) at 10 ng/ml is addedthe next day, and 30 IU/ml human IL-2 (Proleukin; Chiron Corp.) is addedon day two. Seven and 14 days after the primary stimulation, CD8+ Tcells are restimulated with irradiated adherent cells pulsed with 10μg/ml peptide in the presence of 3 μg/ml β2m. Human IL-10 (10 ng/ml) andIL-2 (50 IU/ml) are added 24 and 48 hours later, respectively. Sevendays after the second restimulation, individual wells from the culturesare tested for peptide specific cytotoxicity on peptide loaded T2 cellsin the presence of cold K562 cells (hot/cold target ratio 1:33 ratio).

CTL are also generated from glioblastoma patients. PBMCs from a total of30 HLA-A2+ glioma patients are evaluated for their in vitroresponsiveness against wild-type and superagonist peptides. Theproportion of human patients that will develop specific CTLs capable ofrecognizing the wild-type CD133 peptide after stimulation with thesuperagonist peptide is determined. It is also determined whether theseCTL recognize peptide-pulsed T2 cells or HLA-A2+ cancer stem cell linesthat express CD133.

Intracellular production of IFN-γis detected. A total of 5×10⁴ T cellsare incubated with 10⁵ peptide-loaded T2 cells or with 10⁵ tumor cellsin the presence of 20 μg/ml Brefeldin A at 37° C. Six hours later, thecells are stained with phycoerythrin-conjugated anti-CD8 mAb (CaltagLaboratories, Burlingame, Calif.) in PBS for 25 minutes at 4° C. andfixed with PBS 4% Paraformaldehyde (Sigma). The cells are thenpermeabilized with PBS+0.5% BSA+0.2% saponin (Sigma) and stained withadenomatous polyposis coli-conjugated anti-IFN-γ mAb (PharMingen,Mississauga, Ontario, Canada) for 25 minutes at 4° C. Cells are analyzedon a BD FACSCalibur™ flow cytometer (Becton Dickinson, Mountain View,Calif.).

Enzyme-linked immunosorbent spot (ELISPOT) assay kits (R & D Systems,Minneapolis, Minn.) are used to detect immune responses. Responder (R)1×10⁵ patients' PBMC from before and after vaccination are plated in96-well plates with nitrocellulose membrane inserts coated with captureAb. Stimulator (S) cells (T2 pulsed potential peptide) are added at theR:S ratio of 1:1. After a 24-hour incubation, cells are removed bywashing the plates 4 times. The detection Ab is added to each well. Theplates will be incubated at 4° C. overnight, and the washing steps arerepeated. After a 2-hour incubation with streptavidin-alkalinephosphatase, the plates are washed. Aliquots (100 μl) of BCIP/NBTalkaline phosphatase substrate solution are added to each well todevelop the spots. The reaction is stopped after 60 minutes by washingwith water. The spots are scanned and counted with computer-assistedimage analysis (Cellular Technology Ltd, Cleveland, Ohio). Whenexperimental values are significantly different from the mean number ofspots against non-pulsed T2 cells (background values), as determined bya two-tailed Wilcoxon rank sum test, the background values aresubtracted from the experimental values. This assay provides acoefficient of variation of intra-assay for ELISPOT of less than 10%.

The superagonist-induced CTLs possess higher avidity, due to eitherhigher affinity or stability between TCRs and peptide-MHC complexes. Thehigher avidity correlates with the avidity of T cell-target interactionsand the antitumor responsiveness of T cells. The intensity (Yee et al.,1999, J. Immunol., 162:2227-34), or stability (Dutoit et al., 2002, J.Immunol., 168:1167-71) of specific T-cell staining with HLA tetramers,and threshold of positive staining using titrating doses of tetramers(Ercolini et al., 2005, J. Exp. Med., 201:1591-1602) are indicative ofthe relative avidity of specific T cells.

Example 8 Functional Assays of Immunogenic Peptides

This example demonstrates the usefulness of some of the HLA-A2immunogenic peptides described herein in functional assays.

HLA-A2 positive PBMC isolated by leukapheresis from two healthy humansubjects were obtained from HemaCare Corporation (Van Nuys, Calif.). ThePBMC from each subject were used to prepare CD8+ T cells and autologousdendritic cells for stimulation with immunogenic peptides. Followingstimulation, the T cells were subjected to functional assays.

CD8+ T cells were prepared from each subject's PBMC by positiveselection using CD8 MicroBeads (Miltenyi Biotec, Bergisch Gladbach,Germany), according to the manufacturer's instructions.

Autologous DC were prepared from adherent PBMC (2×10⁶ cells/ml) culturedfor 5 days in the presence of 500 IU/ml granulocyte macrophagecolony-stimulating factor (Leucomax; Schering-Plough, Kenilworth, N.J.)and 500 IU/ml IL-4 (R&D Systems, Minneapolis, Minn.) in complete medium[RPMI 1640 supplemented with 10% heat-inactivated human AB serum, 2 μML-glutamine (Invitrogen) and antibiotics]. On day 5, DC maturation wasinduced by addition of 50 ng/ml TNF-α. On day 7, the mature DC werecollected and pulsed with 20 μg/ml peptide for 4 hours at 20° C.Peptide-pulsed DC not used immediately were frozen for later use.

The peptide pulsed matured DC were diluted to 3×10⁵ cells/mL in CTLcomplete medium and irradiated at 2800 Rads. The initial in vitrostimulation was performed at a DC:T cell ratio of 1:5 by plating 100μL/well of each batch of DC into a 96 well round bottom tissue cultureplate (3×10⁴ DC/well). One plate was prepared per peptide. To all wells,100 μL of the CD8+ enriched T cell population was added at 1.5×10⁶cells/mL was added, resulting in a final concentration of 1.5×10⁵T-cells/well. Plates were incubated at 37° C., 5% carbon dioxide, andhigh humidity.

On days 7 and 14, cryopreserved peptide pulsed mature DC were thawed andused to restimulate the T-cells. Cryopreserved samples of each peptidepulsed DC batch were removed from liquid nitrogen and thawed rapidly ina 37° C. water-bath. Samples were diluted at least 1:10 with AIM Vmedium (Invitrogen, Carlsbard, Calif.). Samples were washed twice bycentrifugation at 400 g for 7 minutes, and representative samples wereevaluated for cell concentration and viability. Each DC batch wasdiluted to 3×10⁵ cells/mL in CTL complete medium containing 20 U/mLIL-2, 10 ng/mL IL-7, and 2 μg/mL of the specific peptide, and the DCwere irradiated with 2800 Rads. In vitro stimulation plates were removedcarefully from the incubator and 90 μL of supernatant (no cells) wereremoved from each well. To each well, 100 μL of the appropriate DC batchwas added. Plates were then incubated further at 37° C., 5% carbondioxide, and high humidity.

After the second in vitro stimulation, the plates were checked daily forT-cell proliferation. When required, plates were split (one plate willbecome two plates) to avoid overgrowth of the CTL. Generally, thisoccurred on days 19, 23, 26, and 30. The plates were mixed to ensure ahomogenous population, then 90 μL of each CTL well was transferred intoa new 96 well U bottomed plate. When splitting was performed within 3days of the last in vitro stimulation, 100 μL of CTL complete mediumcontaining 20 U/mL IL-2 and 10 ng/mL IL-7 was added. If splitting wasperformed later than 3 days from the last in vitro stimulation, 100 μLof complete CTL medium containing 40 U/mL IL-2 and 20 ng/mL IL-7 wasadded. Split plates were returned to the incubator at 37° C., 5% carbondioxide, and high humidity.

On day 21, the T cells were stimulated a fourth time. The processdescribed for in vitro stimulations 2-3 was repeated with the exceptionthat IL-2 was replaced with 25 ng/mL IL-15.

Stimulated CTL were assayed for staining using peptide-HLA-A2 dimers.Cells were collected 19 days after the third stimulation (subject 1) or6 days after the fourth stimulation (subjects 1 and 2). Dimers of eachpeptide presented on HLA-A2 were prepared using BD™ DimerX I:Recombinant Soluble Dimeric Human HLA-A2:Ig Fusion Protein (BDBiosciences) according to the manufacturer's instructions at a 640-foldexcess of peptide and 2 μg of HLA-A2:Ig protein. The HLA-A2:Ig fusionprotein consists of three extracellular major histocompatibility complex(MHC) class I HLA-A2 domains that are fused to the VH regions of mouseIgG1. Immunofluorescence staining was performed essentially according tothe manufacturer's instructions. The CTLwere washed and resuspended inFACS staining buffer. Non-specific binding was blocked with polyclonalhuman IgG. Peptide-loaded HLA-A2:Ig protein was added, and the sampleswere incubated for 60 minutes at 4° C. The cells were washed andblocking solution of human IgG was added again. For immunofluorescence,FACS buffer containing PE-conjugated anti-mouse IgG1 was added.Following incubation at room temperature, the cells were fixed in 2%paraformaldehyde and analyzed by flow cytometry. The percentages ofcells that stained positive (above background) for both dimer and CD8are indicated in Table 7 below. SEQ ID NOs: 11 and 21 stained stronglyabove background, SEQ ID NOs: 28 and 29 stained moderately abovebackground, and the SEQ ID NO:20 staining was indistinguishable frombackground.

TABLE 7 Peptide Dimer Staining SEQ Subject 1 Subject 2 ID NO  AntigenSequence 3rd stim 4th stim 4th stim 11 CD133 YLQWIEFSI 38.64 20.74 19.3921 CD133 FLLPALIFA 20.43 13.23 21.18 28 CD133 ILSAFSVYV 4.15 0.12 14.0820 CD133 GLLERVTRI 0.27 −0.41 −0.32 29 Mart-1a ELAGIGILTV 0.50 0.82 0.68no peptide NT 0 0.01

CTL were collected 6 days after the third and fourth stimulations foranalysis by ELISPOT. Effector cells (5000 or 25,000 T cells stimulatedwith peptide-pulsed DC or DC without peptide) were plated in 96-wellplates with nitrocellulose membrane inserts coated with anti-human IFN-γAb. Stimulator cells (1×10⁵ peptide-pulsed DC) were added to each well.5 μg/ml PHA was used as a positive control. After a 16-20-hourincubation, the cells were removed by washing the plates. A biotinylatedanti-human IFN-γ detection Ab (Mabtech mAb 7-B6-1 Biotin) was added toeach well, and the plates were incubated at 20° C. for 2 hours. After a1-hour incubation with avidin-phosphatase complex (Vectastain ABCElite), the plates were washed. Aliquots of 3-amino-9-ethylcarbazole(AEC) substrate (Vectastain AEC Kit) were added to each well to developthe spots. The reaction was stopped after 4-10 minutes (when spotsappeared) by washing with water. The spots were inspected visually.Wells that clearly had more spots than the negative control wereindicated as positive (Table 8). For subject 1, only Mart-1a waspositive; for subject 3, both Mart-1a and SEQ ID NO:11 were clearlypositive.

TABLE 8 ELISPOT Assay SEQ Subject 1 Subject 2 ID NO Sequence 3rd stim4th stim 3rd stim 4th stim 11 YLQWIEFSI − − − ++ 21 FLLPALIFA − − − − 28ILSAFSVYV − − − − 20 GLLERVTRI − − − − 29 ELAGIGILTV − ++ ++ ++

This example demonstrates that the immunogenic peptides disclosed hereincan stimulate induction of CTL.

OTHER EMBODIMENTS

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. An immunogen of 100 amino acid residues or fewer that elicits acytotoxic T lymphocyte response against HLA-A2 expressing cells thatpresent a CD133 epitope, wherein the immunogen comprises the amino acidsequence of SEQ ID NO:11 with two or fewer amino acid substitutionswithin SEQ ID NO:11.
 2. A composition comprising the immunogen ofclaim
 1. 3. The composition of claim 2, further comprising an adjuvant.4. A composition comprising the immunogen of claim 1 linked to animmunogenic carrier.
 5. The composition of claim 3, further comprising acytokine.
 6. The composition of claim 2, further comprising a liposome,an immuno-stimulating complex (ISCOM), or a slow-releasing particle. 7.The composition of claim 2, wherein the composition comprises antigenpresenting cells.
 8. A method of immunization comprising: administeringto a subject the composition of claim 2 in an amount effective tostimulate an immune response.
 9. An in vitro method for inducing acytotoxic T lymphocyte (CTL) that is specific for a tumor cellexpressing HLA-A2, the method comprising contacting a precursor CTL withthe immunogen of claim 1 under conditions that generate a CTL responseto said tumor cells.
 10. A kit comprising the immunogen of claim
 1. 11.The immunogen of claim 1, wherein the immunogen is 50 amino acidresidues or fewer in length.
 12. The immunogen of claim 1, wherein theimmunogen is 40 amino acid residues or fewer in length.
 13. Theimmunogen of claim 1, wherein the immunogen is 30 amino acid residues orfewer in length.
 14. The immunogen of claim 1, wherein the immunogen is20 amino acid residues or fewer in length.
 15. The immunogen of claim 1,wherein the immunogen is 15 amino acid residues or fewer in length. 16.The immunogen of claim 1, wherein the immunogen is 10 amino acidresidues or fewer in length.
 17. The immunogen of claim 1, wherein theimmunogen is 9 amino acid residues in length.
 18. An isolated peptideconsisting of the amino acid sequence set forth in SEQ ID NO:11 withthree or fewer amino acid substitutions within SEQ ID NO:11.
 19. Theisolated peptide of claim 18, wherein the peptide consists of the aminoacid sequence set forth in SEQ ID NO:11.
 20. The isolated peptide ofclaim 18, wherein the peptide is linked to any of: an immunogeniccarrier, a Toll-like receptor agonist, an immunogenic peptide known tostimulate a T helper cell type immune response, a cytokine, an antibody,a receptor ligand, a lipid, a multiple antigenic peptide, polyethyleneglycol, a leader sequence, a secretory sequence, or a sequence employedfor purification of the peptide.
 21. The composition of claim 3, whereinthe adjuvant is selected from the group consisting of complete Freund'sadjuvant, incomplete Freund's adjuvant, Montanide ISA-51, Lag-3,aluminum phosphate, aluminum hydroxide, alum, and saponin.
 22. Thecomposition of claim 5, wherein the cytokine is selected from the groupconsisting of IL-1, IL-2, IL-7, IL-12, IL-13, IL-15, TNF, SCF, GM-CSF,TLR-3, TLR-4, TLR-7, and TLR-9.
 23. An immunogen of 50 amino acidresidues or fewer that elicits a cytotoxic T lymphocyte response againstHLA-A2 expressing cells that present a CD133 epitope, wherein theimmunogen comprises the amino acid sequence of SEQ ID NO:11 with threeor fewer amino acid substitutions within SEQ ID NO:11.
 24. The immunogenof claim 23, wherein the immunogen is 20 amino acid residues or fewer inlength.
 25. The immunogen of claim 23, wherein the immunogen is 9 aminoacid residues in length.
 26. The immunogen of claim 23, wherein theimmunogen is linked to any of: an immunogenic carrier, a Toll-likereceptor agonist, an immunogenic peptide known to stimulate a T helpercell type immune response, a cytokine, an antibody, a receptor ligand, alipid, a multiple antigenic peptide, polyethylene glycol, a leadersequence, a secretory sequence, or a sequence employed for purificationof the peptide.